The pH of the cytoplasm (pH_i) measured with pH-sensitive microelectrodes in cleavage-stage blastomeres of amphibian (Ambystoma) and teleost (Fundulus) embryos is about 7.7. In electrotonically coupled cell pairs, junctional conductance is rapidly and reversibly reduced by acidification of the cytoplasm. The relation between junctional conductance and pH_i is the same for increasing and decreasing pH and is independent of the rate of change over a wide range. The relation is well fitted by a Hill curve with K = 50 nM (pK= 7.3) and n = 4 to 5. The closure of gap junction channels at low pH_i appears to be a cooperative process involving several charged sites. The absence of hysteresis and identity of effects for fast and slow pH_i changes implies that protons act directly on the channel macromolecules and not through an intermediate in the cytoplasm.