Formamide-induced osmotic shock has been used to detubulate isolated adult rat ventricular myocytes (i.e., disrupt the surface membrane-T tubule junction). Cell volume, calculated from cell length and width, rapidly decreased and increased upon application and removal of formamide, respectively. After treatment with formamide, membrane capacitance decreased by 26.4% (from 199.4 +/- 18.7 pF in control cells to 146.7 +/- 6.4 pF in formamide-treated cells; n = 13, P < 0.05). However, the amplitude of the L-type Ca(2+) current (I(Ca)) decreased by a greater extent (from 0.75 +/- 0.14 to 0.18 +/- 0.03 nA; n = 5, P < 0.05) so that the density of I(Ca) decreased by 74.5%. Simultaneous measurements of I(Ca) and Ca(2+) transients (monitored using fura 2) showed that both decreased rapidly upon removal of formamide. However, the Ca(2+) content of the sarcoplasmic reticulum showed little change. Cross-striations, visualized with the fluorescent dye di-8-aminonaphthylethenylpyridinium, were sparse or absent in cells that had been treated with formamide, suggesting that formamide can successfully detubulate cardiac cells and that I(Ca) is concentrated in the T tubules, which therefore play an important role in excitation-contraction coupling.