The effect of SV40 viral transformation of human fibroblasts on intercellular gap junctional communication (IJC) was investigated using a short-term quantitative assay. IJC was measured using metabolic cooperation in a coculture system of argininosuccinate synthetase- and argininosuccinate lyase-deficient human fibroblasts. These cell lines were transformed with origin-defective adenovirus/SV40 recombinant virions, and IJC was determined both between transformed cells (homologous IJC) and between transformed and untransformed cells (heterologous IJC). At equivalent cell densities, homologous IJC between transformed cells was reduced to 25-55% of the level between untransformed cells. Intermediate levels of IJC (50-70% of normal) were observed in heterologous cocultures of transformed with untransformed cells. Transformed and untransformed cells were equally sensitive to inhibition of IJC by phorbol esters and by glycyrrhetinic acid, and also did not differ in the degree of upregulation of IJC by forskolin. We conclude that SV40 transformation of human fibroblasts leads to a partial impairment of IJC which is additive when both communicating partners are transformed.