The product of the viral src gene (v-src) is the protein tyrosine kinase pp60(v-src). Among the known consequences of pp60(v-src) activity is the reduction in permeability of gap junctions, an effect that is counteracted by the calcium antagonist TMB-8 (8-N,N-[diethylamino]octyl-3,4,5-trimethoxybenzoate). We show here that a decrease in intracellular pH (pH(i)) also counteracts the v-src effect: junctional permeability of cells containing active v-src kinase rose with decreasing pH(i) in the range 7.15 to 6.75, whereas junctional permeability of cells containing inactive v-src kinase or no v-src at all was insensitive to pH in that range. Low pH also counteracted the known action of diacylglycerol on junction, but only when pp60(v-src) kinase was inactive. Immunoblots of whole-cell lysates using an antibody against phosphotyrosine show that phosphorylation on tyrosine of at least one cellular protein, specific for pp60(v-src) kinase activity, was reduced by low pH but not by TMB-8. These results suggest that TMB-8 does not inhibit v-src action on junctional permeability by interfering with tyrosine phosphorylation of a protein crucial for closure of gap junction channels, but that the inhibition by low pH may be via this mechanism.