Possible regulatory effects on cell-to-cell communication of a synthetic diacylglycerol, an activator of protein kinase C (PKC), were examined in pairs of synchronously beating ventricular myocytes of neonatal rats in primary culture. Junctional communication was estimated by measuring either the rate constant of dye diffusion, with the fluorescence recovery after photobleaching technique, or the cell-to-cell electrical conductance with a double whole-cell voltage clamp. The addition of a freshly prepared emulsion of 1-oleoyl-2-acetyl-sn-glycerol (OAG, 100 micrograms/ml), either in the bath or in the solution filling the patch pipet, was seen to interrupt intercellular communication within approximately 8 to 10 min. This effect is neither mimicked by stimulation of PKC by a phorbol ester, nor prevented by PKC inhibitors, making it unlikely that, in these cells, PKC activation could induce intercellular uncoupling. During OAG exposures, the intracellular calcium concentration was very modestly increased (by a factor 1.5 to 2), which does not suffice to account for uncoupling. OAG might trigger interruption of cell-to-cell communication by a mechanism analogous to that of other lipophilic molecules (such as aliphatic alcohols or long chain unsaturated fatty acids) which interfere with gap junctions.