Our laboratory has found that Chinese hamster ovary (CHO) and mouse Y1 adrenocortical carcinoma PKA mutants with a defective R subunit, but not altered C subunits, exhibit increased resistance to cisplatin as well as other DNA-damaging agents. The mechanism of resistance may be associated with increased recognition of the cisplatin-damaged DNA and protein binding to the DNA lesion, thus enhancing DNA repair in the RI alpha mutants. These data suggest that mutation of RI alpha may confer resistance to cisplatin by affecting DNA repair activity. In the present study, we overexpressed RI alpha in human ovarian carcinoma A2780 cells to demonstrate that RI alpha can modulate cellular sensitivity to cisplatin. Retroviral-infected A2780 cells overexpressing wild-type RI alpha cDNA displayed a four- to eightfold greater sensitivity to cisplatin compared with parental cells. Overexpression of RI alpha in the CP70 cisplatin-resistant derivative of A2780 also increased the sensitivity of these cells to cisplatin. Therefore, enhanced expression of the RI alpha subunit of PKA sensitizes cells to the cytotoxic effects of this DNA-damaging agent. These data suggest that RI alpha may act directly, independent of the C subunit, to influence cellular sensitivity to cisplatin. Therefore, modulation of RI alpha expression or its functional status by pharmacological agents may potentially reverse cisplatin resistance in tumors.