Abstract-We have characterized modulation of I Ca by Ca 2ϩ at the t-tubules (ie, in control cells) and surface sarcolemma (ie, in detubulated cells) of cardiac ventricular myocytes, using the whole-cell patch clamp technique to record I Ca . I Ca inactivation was significantly slower in detubulated cells than in control cells (27.1Ϯ7.8 ms, nϭ22, versus 16.4Ϯ7.9 ms, nϭ22; PϽ0.05). In atrial myocytes, which lack t-tubules, I Ca inactivation was not changed by the treatment used to produce detubulation. In the presence of ryanodine or BAPTA, or when Ba 2ϩ was used as the charge carrier, the rate of inactivation was not significantly different in control and detubulated cells. Frequency-dependent facilitation occurred in control cells but not in detubulated cells, and was abolished by ryanodine. These results suggest that Ca 2ϩ released from the SR has a greater effect on I Ca in the t-tubules than at the surface sarcolemma. This does not appear to be due to differences in local Ca 2ϩ release from the SR, because the gain of Ca 2ϩ release was not significantly different in control and detubulated cells. These data suggest that the t-tubules are a key site for the regulation of transsarcolemmal releases. 2 The transverse (t-) tubules of ventricular myocytes play an important role in this process. These tubules are invaginations of the sarcolemma that occur at the Z-line, perpendicular to the longitudinal axis of the cell (see review). 3 Functional and immunohistochemical data suggest that I Ca occurs predominantly in the t-tubules, adjacent to RyRs, which are also located predominantly at the t-tubules. 4 -6 Thus, it appears that the t-tubules are the major site of Ca 2ϩ entry and hence Ca 2ϩ release in cardiac ventricular myocytes. Conversely, Ca 2ϩ released by the SR can modulate I Ca ; this plays an important role in cellular Ca 2ϩ homeostasis, controlling Ca 2ϩ entry via negative feedback. 7,8 However, it is unknown whether the efficacy of coupling between SR Ca 2ϩ release and I Ca is the same in the t-tubule and surface membranes, so that the relative importance of these sites in cellular Ca 2ϩ homeostasis is unknown. We have, therefore, investigated the regulation of I Ca by Ca 2ϩ released from the SR in normal ventricular myocytes, in which I Ca triggers Ca 2ϩ release predominantly at the t-tubules, and in myocytes in which the t-tubules have been physically and functionally uncoupled from the surface membrane (detubulated), 5 in which Ca 2ϩ release occurs predominantly at the surface membrane. 9