Transverse tubules (TT), plasmalemma invaginations perpendicular to the long axis of the cardiomyocyte, facilitate rapid action potential transmission to the cell interior and efficient cardiac excitation-contraction coupling (ECC). TTs have also been noted to branch in longitudinal directions, such structures being termed transverse-axial tubules (TATs). Tubule alteration, and changes in expression of proteins important for normal ECC, have been noted in heart disease[1]. We explored the relationship between ECC-related protein expression and TT/TAT structure during pre- and post-natal cardiac development to adulthood as this may be informative for understanding pathological maladaptation that involve reversion to fetal molecular phenotypes. Hearts were collected from guinea pigs, killed according to Home Office licensed regulations, at developmental stages: fetal (between gestation days (G) 55-68; term=G67-68); neonatal weeks one(NW1), two (NW2) and three (NW3); and adult. Excised hearts were flushed with cold cardiologic solution and (i) left ventricles (LV) frozen for subsequent protein analysis by western blotting or (ii) retrogradely perfused with fixative and LV processed for ultrastructural examination by serial block face-scanning electron microscopy. Values are mean ± SEM, compared by one-way Anova and Bonferroni posthoc test (p<0.05). Expression of β2 adrenoceptor, a putative TT marker, increased in adults (0.87±0.14-fold relative to positive control) and later neonates NW2(0.90 ±0.04-fold) compared to fetal G55/57 (0.44±0.04-fold) or early neonates NW1 (0.5±0.04-fold). Junctophilin2, a determinant of TT integrity, was expressed in G55/57 and, surprisingly, was invariant among the biological groupings. Expression of Cavin1, a general plasmalemmal marker, increased through fetal and neonatal development to adult. In contrast, flotillin2 expression was unchanged between groupings. Analysis of digitally reconstructed (Amira 6.0) serial EM images revealed developmental changes in cardiomyocyte structure. Sarcomere length narrowed from G55/57 (2.28±0.01 µm) to NW1 (1.90 ±0.01µm) and adult (1.92 ±0.01 µm; assessing 250 sarcomeres from 15 regions of interest). TTs were observed at G58-59 although longitudinal tubular elements predominated. Total tubular surface area (30 serial sections, n=6 for each condition), increased from 0.68±0.08% at G64/68 to 2.54±0.48% in adult. TT diameter (100-150 serial sections, >50 TTs) was 0.27±0.02 µm in G65/68 and 0.40 ±0.02 µm in adult.Fetal cardiac TT/TATs as early as the mid-third trimester of guinea pig pregnancy. Changes in TT/TAT abundance through pre- and post-natal development to adulthood mirror the changes in expression of some (β2 adrenoceptor, cavin1) but not other (junctophilin2) proteins likely to be important for maturation of ECC