In vertebrate embryos gap junctional conductance (gj) is reduced by transjunctional voltage (Vj) and by cytoplasmic acidification; in each case sensitivity is comparable to those of other channels gated by voltage and ligand-receptor binding. We show here that the mechanisms by which Vj and intracellular pH (pHi) gate gj are apparently independent. Partial reduction of gj by lowering pHi neither attenuates nor enhances further reduction by Vj. Certain drugs irreversibly (glutaraldehyde, 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) or reversibly (retinoic acid) abolish dependence of gj on pHi without appreciably affecting kinetic properties of voltage dependence or the shape of the steady-state Vj-gj relation. These findings suggest that the mechanisms by which pHi and Vj act on the gap junction are at least partially distinct and presumably involve separate regions of the junctional macromolecules.