Junctional permeability in normal and transformed NRK cells was quantitatively assessed by microinjecting fluorescent dye into one cell of a pair, digitizing the changes of fluorescence intensity using video analysis techniques, and applying the digital values to a solution of Fick's diffusion equation. We show that this approach reliably estimates the junctional permeance of a cell pair. Cells that are temperature sensitive for transformation were shown to also be temperature sensitive vis-ă-vis junctional permeance. Thus permeance values were reduced approximately 80-90% on transformation by either a mutant Rous sarcoma virus or a mutant Moloney murine sarcoma virus. Cells transformed by wild-type Kirsten sarcoma virus were also shown to possess levels of junctional permeance significantly lower than nontransformed controls. The transformed junctional phenotype could be observed as early as 15 min after shifting to transformation-permissive conditions. Our results suggest that the oncogenes src, ras, and mos exert their effects on NRK cell junctions via converging pathways, of which one may be phosphorylation of junctional proteins.