Methotrexate (MTX)(2 x 10(-8) M) inhibited DNA synthesis in CCRF-CEM cells, causing cells to accumulate in early S phase while cellular RNA content and cell size continued to increase. Two-parameter flow cytometric analysis of DNA and RNA showed these cells to be unbalanced with excessive RNA relative to DNA content. Fifty % of cells remained viable after a 96-hr exposure to 2 x 10(-8) M MTX. In contrast, 10(-4) M MTX inhibited cell cycle progression of cells in both G1 and S phases and also prevented the development of unbalanced growth. In this instance, cell viability was reduced to 10% after 96 hr of drug exposure. The relative contribution of inhibition of thymidylate and purine biosynthesis to MTX cytotoxicity was investigated by addition of exogenous thymidine (10(-5) M) or hypoxanthine (10(-4) M). Thymidine reduced the cytotoxicity and inhibition of DNA synthesis caused by both doses of MTX and prevented classical unbalanced growth with 2 x 10(-8) m MTX; treatment with 10(-4) M MTX resulted in a form of unbalanced growth where cells had a relative excess of DNA compared with RNA content. The addition of hypoxanthine enhanced the classical unbalanced growth pattern seen with 2 x 10(-8) M MTX but was accompanied by a partial reduction of both the MTX-induced cytotoxicity and the inhibition of DNA synthesis (to an extent similar to that seen with exogenous thymidine). Potentiation of cell killing was observed with the addition of hypoxanthine to cells treated with 10(-4) M MTX. Complete rescue from MTX cytotoxicity at both concentrations was found only when both thymidine and hypoxanthine were present. These finding suggest that MTX cytotoxicity is associated with inhibition of DNA synthesis resulting from the disturbance of both thymidylate and purine biosynthesis.