The influence of heptanol on gap junctional and non-junctional membrane currents was studied in cultured neonatal rat heart cells using both the whole cell and perforated patch voltage-clamp method. With both methods, exposure to heptanol produced a dose-dependent decrease in the junctional current (dissociation constant = 0.54 and 1.20 mM for whole cell and perforated patch experiments, respectively). Heptanol-induced uncoupling was reversible. In the same concentration range, heptanol reduced all nonjunctional membrane ionic currents examined. This suggests that heptanol does not act specifically on gap junction channels but rather on the structure of the lipid membrane. This hypothesis is strengthened by the observation that in monolayer cultures of neonatal rat heart cells fluorescence steady-state anisotropy decreased proportional with increasing the heptanol concentration in the bath. Single-channel conductances (gamma j) were identical with both recording methods (21 and 40-45 pS); heptanol did not alter gamma j. Under conditions of reduced junctional coupling induced by heptanol, junctional conductance (gj) displayed voltage sensitivity at values of gj at which no voltage sensitivity could be observed under control conditions. These results suggest that heptanol-dependent uncoupling was caused by a decrease in open probability of the gap junction channels.