Phorbol 12-myristate 13-acetate (TPA) increases the number of colonies surviving methotrexate (MTX) exposure in a dose-dependent manner upon short incubation with Chinese hamster ovary (CHO) cells. Seventy percent of the isolated colonies showed increased copy number for the dihydrofolate reductase gene. EGTA prevents the increase in resistance triggered by TPA. Calcium ionophore A23187 and angiotensin II also increase this resistance, suggesting that calcium is involved in this process. Protein kinase C (PKC) from CHO cells is rapidly activated by TPA, A23187 and angiotensin II. PKC inhibitors, 1-(5-Isoquinolinylsulphonyl)-2-methyl-piperazine (H-7), glycyrrhetinic acid, staurosporine and calphostin C decrease the generation of resistant colonies to MTX upon incubation with TPA. However, 5 nM staurosporine on its own increases resistance to MTX while having the ability to translocate CHO PKC. In vitro, H-7, staurosporine and calphostin C inhibit PKC activity translocated by TPA incubation with CHO cells. We conclude that PKC, the activity of which is dependent on calcium and phospholipids, is part of the pathway that leads to development of increased resistance to MTX. Thus, inhibition of PKC prevents the appearance of this resistance. Our results suggest the possibility of using non-toxic PKC inhibitors as resistance modulators in MTX chemotherapy.