The role of small conductance, calcium-activated potassium (SK) channels in atrial repolarisation has been debated due to contradictory effects of SK channel inhibitors.  We proposed that the native SK current that contributes to atrial repolarisation is carried by a heteromer comprised of both SK2 and SK3 subunits. This heteromer displays a unique but poorly understood pharmacology (Hancock et al., 2015).  As cardiac SK channel expression may change in disease states, evaluation of SK channel function in the healthy human myocardium is difficult (Darkow et al., 2021).  Consequently, this study was undertaken to evaluate the use of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a potential model system to probe human SK channel composition and function. Human iPSCs (REBL-PATs; a gift from Professor Chris Denning: Mosquiera et al., 2018) were differentiated in the presence or absence of retinoic acid, to produce atrial (Atr-hiPSC-CM) or mixed atrial/ventricular (AV-hiPSC-CM) lineages, and cultured in 2D monolayers for up to 90 days before dissociation onto coverslips. Experimentation occurred at three separate timepoints (1: day 35±5; 2: 65±5 & 3: 95±5). Whole-cell voltage clamp was used to evaluate single-cell responses to the SK channel inhibitor UCL1684 at room temperature. Additionally, the presence of acetylcholine (ACh)-activated K+ current (IK,ACh) was probed as an atrial-selective response.  Immunocytochemistry was used to evaluate expression of MLC2v, a ventricular-specific myosin light chain isoform; and TNNI3, a troponin isoform absent in the foetal heart (Lee et al, 2017). Finally, whole-cell current clamp was performed to measure maximum diastolic potentials (37°C).  Data are presented as mean ± S.E.M.  Initial patch clamp experiments on Atr-hiPSC-CMs at timepoints 1 and 2 of culture showed that UCL1684 had no effect on membrane current evoked during a voltage ramp from -100mV to +100mV (n=6 & 5 respectively) despite an increased magnitude and incidence of IK,ACh in response to 1µM ACh (1.46±0.40pA/pF in 88% of Atr-hiPSC-CMs vs. 0.50±0.18pA/pF in 38% of AV-hiPSC-CMs; n=8 for each; p<0.05, Student’s t-test) and the absence of the ventricular marker MLC2v (1% positive Atr-hiPSC-CMs vs. 29% AV-hiPSC-CMs).  The immature phenotype of hiPSC-CMs is an established limitation of their use (Feyen et al., 2020).  Prolonging time in culture (to timepoint 3) led to a more mature cell phenotype, significantly increasing the expression of TNNI3 and hyperpolarising the maximum diastolic potential (-54.4±1.7mV [timepoint 1, n=13] vs. -61.6±2.3mV [timepoint 2, n=8] vs. -74.2±2.2mV [timepoint 3, n=4]; p<0.001, one-way ANOVA). Prolonged culture also decreased the spontaneous action potential firing rate of single cells over the 3 timepoints (2.7±0.2Hz [~35, n=13] vs. 0.9±0.2Hz [~65, n=8] vs. 0Hz [~95, n=4]; p<0.0001, one-way ANOVA).  Extending culture time to timepoint 3 resulted in a proportion of Atr-hiPSC-CMs (3 of 7 cells) responding to SK channel inhibition (1.80±1.0pA/pF at +80mV).  In conclusion, although a UCL1684-sensitive current could not be recorded consistently from Atr-hiPSC-CMs cultured under these conditions, our data suggest a potential link between cell maturity and UCL1684 responsiveness.   Acknowledgement:  This work was funded by the British Heart Foundation (FS/17/60/33474).