In this study, we compared the rate of detubulation of adult mouse and rat ventricular myocytes over a 72 h culture period. The T-tubule density was measured in the following two ways: (i) as whole-cell capacitance in voltage-clamped myocytes relative to cell area; and (ii) using di-8-ANEPPS staining and confocal microscopy. In adult rat ventricular myocytes, whole-cell capacitance/area was significantly reduced from 47 ± 3 fF μm2 (mean ±s.e.m.; n= 16) in freshly isolated (control) cells to 36 ± 2 fF μm2 (n= 20) after 72 h in culture. The T-tubular density, as assessed optically using di-8-ANEPPS staining, at 48 h was significantly reduced to 70 ± 7% (n= 14) compared with control cells. The T-tubular density was further reduced after 72 h in culture to 43 ± 7% (n= 10) compared with control cells. In contrast, in mouse myocytes neither whole-cell capacitance relative to cell area nor optical assessment of T-tubules showed any significant reduction in capacitance/cell area or T-tubule density after 72 h of culture. Expression of caveolin-3 (CAV-3) (a marker of T-tubule development) was also measured, and a significant reduction was observed in CAV-3 expression in rat myocytes at 48 (80 ± 5.5%; n= 6) and 72 h (66 ± 9.5%; n= 6) compared with control cells. The expression of CAV-3 in mouse myocytes was not significantly reduced even at 72 h. When rat ventricular myocytes were paced in culture for 72 h they exhibited no significant improvement in T-tubule density or CAV-3 expression compared with non-paced cultured cells. In rat myocytes, sarcomere length shortening was significantly reduced in myocytes cultured for 48 (4.96 ± 0.72%; n= 26) and 72 h (4.32 ± 0.80%; n= 26) compared with freshly isolated cells (7.12 ± 0.56%; n= 18). Mouse myocytes, after 24 h in culture, were unable to follow external pacing. These results suggest that detubulation in quiescent culture is slower in the mouse than the rat and that this loss of T-tubules profoundly affects excitation–contraction coupling in rat myocytes.