Hoechst 33342 stain has been used previously in conjunction with fluorescence activated cell sorting to separate cell populations into the various phases of the cell cycle. The present experiments were designed to evaluate the toxicity of this stain in cells derived from solid KHT sarcomas which were untreated or irradiated prior to dissociation. In both untreated and irradiated cells, stain toxicity increased with increasing exposure times but was always greater in treated cells; for example, after stain exposures of 1 h at 10 microM or 2 h at 5 microM, survival was reduced 25- to 45-fold in irradiated cells and 4- to 5-fold in untreated cells. To determine whether this toxicity occurred equally in all phases of the cell cycle or preferentially in a particular phase, cells derived from untreated or irradiated KHT tumors were separated into the various phases of the cell cycle using centrifugal elutriation. Cells in each phase then were divided in two, one-half being stained with 5 microM Hoechst 33342 for 2 h, the other half being handled in an identical manner but not stained. These particular stain exposure conditions were chosen for detailed evaluation because they provided the best DNA histograms. In both untreated and irradiated cells the cytotoxic effects of Hoechst 33342 were found to be significantly greater in cells in the S phase than in cells in G1 and G2-M phases of the cell cycle. This toxicity, particularly its cell cycle specificity, suggests a potentially severe limitation for the use of Hoechst 33342 in combination with fluorescence activated cell sorting in studies aimed at determining treatment efficacies in various phases of the cell cycle.