The mechanism of synergy between 3'-azido-3'-deoxythymidine (AZT) and anticancer agents was investigated with emphasis on cell-cycle events. Exposure of exponentially growing WiDr human colon carcinoma cells to AZT resulted in synchronization of cells in the S phase of the cell cycle. Following treatment with AZT at 50 or 200 microM, 62% +/- 3% or 82% +/- 4% of the cells were in the S phase as compared with 36% +/- 2% in the control. Bromodeoxyuridine uptake studies revealed that the synchronized cells actively synthesized DNA. At concentrations of up to 200 microM, AZT produced a cytostatic rather than cytotoxic effect as indicated by viability and cell growth measurements. At 200 microM, AZT-induced synchronization was significant (P = < 0.001) after 12 h of drug exposure, reached a maximum at 24 h, and reversed to baseline levels by 72 h even in the continued presence of the drug. This indicates that AZT-induced cytostasis is a transient and reversible effect. The cell-cycle events seen with AZT in WiDr cells were also observed in eight of nine human tumor cell lines tested. Isobologram analysis of WiDr cells preexposed to AZT for 24 h and then exposed to either AZT-5-fluorouracil or AZT-methotrexate for a further 72 h revealed synergy between AZT and the anticancer agents, indicating that AZT-induced synchronization may have therapeutic benefits.