The effect of methotrexate on 125I-iododeoxy-uridine (125IUDR) incorporation into the DNA of L1210 and L1210/MTX ascites cells was determined at different stages of tumor development. Methotrexate administration to drug sensitive L1210 cells during the early phases of tumor growth invariably produced a dramatic decline in precursor utilization. In advanced L1210 ascites populations small drug doses (1 mg/kg) increased 125IUDR incorporation to about twice the control value, while larger doses (10 mg/kg or more) decreased uptake to less than half the original value. Log phase populations of drug resistant L1210/MTX cells resembled terminal L1210 ascites cells in that precursor uptake was enhanced by small amounts of drug and decreased by larger amounts. During the terminal phase of L1210/MTX development all drug levels up to 100 mg/kg produced an increase in 125IUDR incorporation. In parallel studies an attempt was made to correlate the in vivo perturbations of the DNA precursor metabolism to the lethal action of the drug. In terms of cell killing, L1210/MTX cells proved completely resistant to the action of methotrexate, while peritoneal L1210 cells remained fully sensitive throughout the course of ascites growth. Thus, although the results indicated some overall correlation between drug induced inhibition of DNA synthesis and subsequent cell death, the relationship between these 2 parameters would appear to be too complex and ambiguous to be used as a technique for predicting the chemotherapeutic effectiveness of the drug.