Electrical coupling, mediated by gap junctional channels connecting pairs of murine pancreatic acinar cells and Chinese hamster ovary (CHO) cells, was studied using the double whole cell patch clamp technique. Two approaches were used to reduce the junctional conductance (gj) in order to study gj at the single channel level. During spontaneous uncoupling, single channel conductances of 130 pS and 27 pS could be characterized using freshly isolated acinar cells. In most experiments, stepwise conductances could not be discriminated while gj decreased gradually below 10 pS. In CHO cell pairs, discrete junctional channel conductances of 120 pS, 70 pS, 50 pS, 37 pS and 22 pS were identified. Exposure of pancreatic acinar cell pairs to 0.4 mM octanol resulted in rapid and reversible uncoupling. Discrete junctional conductance steps could not clearly be identified down to a gj of about 3 pS. The influence of the composition of the pipette solution on spontaneous uncoupling was investigated. Addition of 5 mM ATP and 0.1 mM cAMP to the pipette electrolyte was sufficient to stabilize coupling in the experimental time range of up to 1 h. Different mechanisms of uncoupling, including an increase of flickering in the channel open state, and modulation of the number of channels exhibiting different conductance or subconductance states are discussed.