Interaction between polyoma virus (Py) encoded middle tumor antigen (MTAg) and the cellular src gene product, pp60c-src, has been shown to be correlated with the enhancement of pp60c-src kinase activity and the transforming ability of the virus. In the present study, FR3T3 cells were transfected with plasmids encoding MTAg and used to study the effects of the antineoplastic agent, doxorubicin (DOX), on pp60c-src kinase activity. Our results showed that transfection of FR3T3 cells with MTAg results in relatively high levels of MTAg expression and enhancement of pp60c-src kinase activity several folds over that observed in the control cells. Treatment of the transformed MTF4 cells with DOX up to 5 x 10(-6) M was shown to have no effect on the in vitro phosphorylation of MTAg and pp60c-src in the drug-treated cell lysates. On the contrary, treatment of src immunocomplexes with DOX resulted in a reduction in kinase activity. The inhibition appears to be a dose-dependent competition with respect to ATP concentrations.