Treatment of human breast (MCF-7) and lung (A549) adenocarcinoma cell lines with 10 micrograms/ml cycloheximide provided substantial protection from paclitaxel-induced cytotoxicity. Addition of cycloheximide to cells at 0, 6, 12 or 18 h into a 24 h exposure to paclitaxel resulted in cytotoxicity similar to that found in cells treated with paclitaxel alone for only 0, 6, 12 or 18 h, respectively. DNA flow cytometry showed that paclitaxel blocked cells in G2/M. Mitotic index studies demonstrated that paclitaxel arrested cells in mitosis and that prolonged exposure to paclitaxel resulted in the development of multiple micronuclei. Concurrent incubation of cells in cycloheximide prevented the development of a G2/M block, mitotic arrest and micronuclei formation. The addition of cycloheximide to cells at 6 or 12 h into a 24 h exposure to paclitaxel reduced the degree of G2/M block to that produced by incubation of cells in paclitaxel alone for only 6 or 12 h. Mitotic index studies confirmed that cells treated with cycloheximide during paclitaxel exposure had a marked reduction in the percentage of cells in mitosis. However, the percentage of paclitaxel-treated cells which had multiple micronuclei was increased in cells treated with cycloheximide. These results indicate that entry into mitosis is a prerequisite for paclitaxel-induced cytotoxicity and that cycloheximide reduces cytotoxicity due to paclitaxel by preventing cells from entering mitosis. However, once cells have entered mitosis in the presence of paclitaxel, protein synthesis is not required for the development of multiple micronuclei and cytotoxicity.