Activity of the thymidylate synthase cycle was compared in the human ovarian carcinoma cell line A2780 and a subline that is resistant to cisplatin by a factor of 3. Resistant cells exhibited a 3-fold increase in mRNA for both dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+-oxidoreductase, EC 1.5.1.3) and thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) when compared with the parent line. Resistance to cisplatin also resulted in a 2.5-fold increase in enzyme activity for dihydrofolate reductase and thymidylate synthase; however, this increase did not result from amplification of the genes for these two enzymes. These data suggest that the initial step of cisplatin resistance in A2780 cells is a consequence of enhanced expression of the thymidylate synthase cycle.