The signal transduction pathway of cAMP, mediated by the cAMP-dependent protein kinase (PKA), is involved in the regulation of metabolisms, cell growth and differentiation and gene expression. Isolated PKA mutants from Chinese hamster ovary (CHO) cells were used in our laboratory to study the role of cAMP in the development of drug resistance in cancer. We have found that PKA mutants harboring a defective regulatory (RI alpha) subunit, but not the catalytic (C) subunit, are more resistant to the chemotherapeutic drug cisplatin. To clarify the role of PKA in cisplatin resistance, we have performed a step-wise selection with a CHO RI alpha subunit mutant cell line, 10248, for further resistance to cisplatin. A representative clone (10248/CDDP(R)-5) was used for further characterization. These cisplatin-resistant PKA mutant cells remained refractory to cAMP-induced growth inhibition and had decreased PKA activity comparable to the parental 10248 mutant cells. Furthermore, 10248/CDDP(R)-5 also exhibited cross-resistance to the nitrogen mustard melphalan but maintained the same sensitivity as wild-type cells to non-DNA-damaging agents such as methotrexate. The mechanism of resistance may be due to increased DNA repair as assessed by the host cell reactivation assay. We speculate that mutation and functional inactivation of PKA may result in deregulated growth response to cAMP, as well as the acquisition of resistance to cisplatin and other DNA-damaging agents in cancer.