The effects of oleic acid (OA) on gap junction-mediated intercellular communication between A7r5 cells and neonatal rat cardiac myocytes were determined. In A7r5 cells the extent of dye coupling was influenced in a biphasic manner by increasing concentrations of OA. Low concentrations of OA (0.1-1 microM) reduced the incidence of dye coupling from 90% (in control cells) to approximately 50%. Further increases in OA concentration, up to 100 microM, had no further effect on extent of dye coupling. In contrast, dye coupling between cardiac myocytes was reduced to near zero levels in a linear fashion by 1-25 microM OA. Whereas high OA concentrations reduce junctional conductance (gj) between heart cells to zero [J. M. Burt, K. D. Massey, and B. N. Minnich. Am. J. Physiol. 260 (Cell Physiol. 29): C439-C448, 1991], gj between A7r5 cells was decreased by a maximum of 45% by OA. These differences in OA sensitivity between the two cell types were not explained by differences in the rate or magnitude of OA uptake by the cells or by differences in the fraction of incorporated OA accessible to albumin washout, i.e., the plasma membrane fraction. Instead, the activity of the individual channel types exhibited different sensitivities to OA. In the presence of increasing concentrations of OA, the activities of first the 70-pS channel population [composed of connexin40 (Cx40)] and then the 108-pS channel population (composed of Cx43) were diminished, leaving predominantly the 140-pS channels (composed of Cx43) at high OA concentrations. The uncoupling effects of OA in both cell types could be reversed by washout with albumin-containing solution; however, higher concentrations of albumin and more vigorous wash conditions were required for full recovery in the A7r5 cells. In addition, albumin also reversed the effects of OA on channel activity. These data suggest that OA binds with greater affinity to the 70- vs. 108- or 140-pS channels and associated with binding is reduced channel activity.