Overexpression of the cellular src gene in NIH-3T3 cells causes reduction of cell-to-cell transmission of molecules in the 400-700 dalton range. This down-regulation of gap junctional communication correlates with the activity of the gene product, the protein tyrosine kinase pp60c-src. The down-regulation is enhanced by point mutation of Tyr527 (a site phosphorylated in pp60c-src and which inhibits kinase activity) or by substitution of the viral- for the cellular-src carboxyl terminal coding region. Mutation of Tyr416 (a site phosphorylated upon Tyr527 mutation) suppresses both the down-regulation by Tyr527 mutation and that by gene overexpression. The regulation of communication by src may be important in the control of embryonic development and cellular growth.