Human embryonic stem cells (hESC) are presently the stem cell type with the greatest proven capacity for producing phenotypically authentic cardiomyocytes, and are therefore of great interest in terms of cardiac repair, or as a model system for cardiac physiology and pathophysiology. Embryonic-like cells can also be induced by expression of “stemness” factors within somatic cells, and these induced pluripotent cells (iPSC) can be differentiated to cardiomyocytes (iPSC-CM) using similar protocols. These raise the possibility of creating cardiomyocytes which are immune matched to the subject, or which harbour a known disease-causing mutation. Based on their gene expression patterns, electrophysiological, morphological and contractile properties, the majority of human embryonic stem cell-derived cardiomyocytes (hESC-CM) or iPSC-CM initially resemble human immature cardiomyocytes, but have the capacity to mature in a number of respects. We have investigated both acute and long-term responses in hESC-CM and iPSC-CM, in comparison to adult human ventricular myocytes. Matching of excitation-contraction coupling between hESC-CM and adult myocytes is of particular concern with respect to the generation of arrhythmias. Delivery of immature myocardium into the mature ventricle has the potential to promote abnormal rhythms both because the hESC-CM have spontaneous contractile activity and because any difference from adult ventricle will produce a heterogeneous substrate. hESC-CM were followed for 5 months after induction of differentiation for beating activity, and the development of excitation-contraction coupling was quantitated by determining the relative contributions to contraction through calcium entry or calcium release from intracellular stores. Transition from the immature to mature cardiac phenotype is characterised by increasing dependence on intracellular calcium. HESC-CM were challenged with either caffeine (rapid application) or thapsigargin, both of which will empty intracellular calcium stores. While hESC-CM before day 46 were insensitive to these manoeuvres, those between day 129-150 showed prolonged relaxation, slow calcium transients and frequent cessation of beating. These were indications of development of the adult phenotype, and were comparable with effects in adult human ventricular cardiomyocytes. Sensitivity to arrhythmic challenge with bile acids (in a model of obstetric choleostatis) altered over a shorter timescale, being largely absent by 71 days. In terms of pharmacological response, stimulatory beta1- and beta2AR pathways in hESC-CM were well matched to adult human ventricular myocytes even at early time points, although the absolute sensitivity to catecholamines was 5-10-fold lower. Inhibitory muscarinic and adenosine responses were also evident, and agonists for these receptors reduced both basal and catecholamine-stimulated chronotropy. Adenosine acted through A1 adenosine receptors with a sensitivity approximately 20-fold less than in adult human ventricular myocytes, but the maximum efficacy increased over 60 days in culture. Parallel increases in muscarinic receptor responses suggest greater activity of IkAch as the key developmental change. Growth and proliferation potential are important for expansion of hESC-CM after implantation. We showed that hESC-CM retain their capacity for increase in size in long-term culture (<180 days post differentiation), although proliferative activity decreases. Exposing hESC-CM to hypertrophic stimuli such as equiaxial cyclic stretch, angiotensin II and phenylephrine (PE) increased cell size and volume, percentage of hESC-CM with organised sarcomere, angiotensinogen and ANF mRNA and immunoreactive levels and cytoskeletal assembly. Changes in cell size by PE were inhibited a range of known anti-hypertrophic agents including p38-MAPK, calcineurin/FKBP and mTOR blockers. Inhibitors of ERK, JNK and CaMKII partially reduced PE-effects; PKG or GSK3 inhibitors had no effect. Modulation of kinase signalling had independent effect on proliferative capacity and cell cycle distribution of the cells, suggesting that cell growth and cell cycle progression are separable processes in hESC-CM. Apoptotic and inflammatory responses were well developed even in early hESC-CM, but innate immune signalling through key Toll receptors was undeveloped even after 4 months of differentiation. Overall, maturation is a multifaceted process, and matching of hESC-CM to the adult phenotype requires consideration of many aspects of long and short-term cellular regulation. These findings have implications for both implantation of hESC-CM/iPSC-CM and their use as a high-fidelity model system.