The retinoblastoma gene product, Rb, has previously been implicated as an obligatory component in the antiproliferative effects mediated by the lipid second messenger, ceramide. We have evaluated both the apoptotic effects and the effects on cell cycle distribution of the exogenous cell-permeable ceramide, N-hexanoyl-D-sphingosine, in an Rb-null human bladder tumor cell line, 5637, as well as in retrovirally infected, Rb(+) clones derived therefrom. These cell lines demonstrated comparable sensitivity to N-hexanoyl-D-sphingosine in a neutral red dye uptake assay. Exposure of the Rb-null parental cell line to 20 microM N-hexanoyl-D-sphingosine for 24 h resulted in a classical pattern of DNA fragmentation that was accompanied by apoptotic nuclear morphological alterations. In contrast, the Rb(+) clones demonstrated suppression of DNA fragmentation in response to N-hexanoyl-D-sphingosine. Similarly, the frequency and degree of alteration of nuclear morphology in Rb(+) cells was also suppressed. Flow cytometric analysis of the parental and infected clones indicated that expression of Rb was without effect on their cell cycle distribution, with or without exposure to N-hexanoyl-D-sphingosine for 25 h; tunel assay confirmed that in this time frame apoptotic cells were far less frequent in the Rb(+) clones than in the parental 5637 cells. Human tumor cell lines derived from three other histological origins, breast and prostatic carcinomas and osteogenic sarcoma, also demonstrated very similar cytotoxic sensitivities to N-hexanoyl-D-sphingosine, irrespective of the expression of Rb. We conclude that Rb is not required for ceramide-induced apoptosis and that Rb can actually inhibit the DNA fragmentation and nuclear morphological changes associated with classical apoptosis.