A clone of human HeLa cells stably transfected with mouse connexin40 DNA was used to examine gap junctions. Two separate cells were brought into physical contact with each other ("induced cell pair") to allow insertion of gap junction channels and, hence, formation of a gap junction. The intercellular current flow was measured with a dual voltage-clamp method. This approach enabled us to study the electrical properties of gap junction channels (cell pairs with a single channel) and gap junctions (cell pairs with many channels). We found that single channels exhibited multiple conductances, a main state (gamma j(main state)), several substates (gamma j(substates)), a residual state (gamma j (residual state)), and a closed state (gamma j(closed state)). The gamma j(main state) was 198 pS, and gamma j(residual state) was 36 pS (temperature, 36-37 degrees C; pipette solution, potassium aspartate). Both properties were insensitive to transjunctional voltage, Vj. The transitions between the closed state and an open state (i.e., residual state, substate, or main state) were slow (15-45 ms); those between the residual state and a substate or the main state were fast (1-2 ms). Under steady-state conditions, the open channel probability, Po, decreased in a sigmoidal manner from 1 to 0 (Boltzmann fit: Vj,o = -44 mV; z = 6). The temperature coefficient, Q10, for gamma j(main state) and gamma j(residual state) was 1.2 and 1.3, respectively (p < 0.001; range 15-40 degrees C). This difference suggests interactions between ions and channel structure in case of gamma j(residual state). In cell pairs with many channels, the gap junction conductance at steady state, gj, exhibited a bell-shaped dependency from Vj (Boltzmann fit, negative Vj, Vj,o = -45 mV, gj(min) = 0.24; positive Vj, Vj,o = 49 mV, gj(min) = 0.26; z = 6). We conclude that each channel is controlled by two types of gates, a fast one responsible for Vj gating and involving transitions between open states (i.e., residual state, substates, main state), and a slow one involving transitions between the closed state and an open state.