Retinoic acid (RA) induces differentiation, followed by apoptosis in acute promyelocytic leukemia (APL) cells, both in vitro and in patients. One problem in understanding these mechanisms is to distinguish molecular events leading to differentiation from those leading to apoptosis. We have identified a leukemic cell line, PLB-985, where RA directly induces apoptosis with no morphologic, genetic, or cell-surface marker evidence of differentiation. These cells differentiate following dimethyl sulfoxide (DMSO), but not RA, treatment. Two-color flow cytometry showed no alteration of the cell cycle after RA treatment, and cell-surface marker analysis of CD11a, CD11b, and CD13 showed no modulation typical of differentiating cells. RNA expression of myeloblastin and transglutaminase, genes regulated by RA-induced differentiation in NB4 cells, was unchanged by RA treatment. Instead, RA induced apoptosis, as shown by typical apoptotic morphological features, genomic DNA laddering, and positive labeling in the TUNEL assay. We found that induction of apoptosis in this model requires a different pattern of retinoid receptor binding and transcriptional activation than is seen in APL cells. As previously described, treatment with retinoid receptor-selective ligands showed that stimulation of RAR alone is sufficient to induce differentiation and apoptosis in NB4 cells, and that stimulation of RXR has no effect on the parameters analyzed. In PLB-985 cells, on the other hand, apoptosis was induced only upon costimulation of both RAR and RXR. Stimulation of either receptor alone had no effect on the cells. Consistent with these findings, bcl-2 RNA and protein levels were downregulated after stimulation of both RAR and RXR, but not with an RAR-specific ligand alone, as in NB4 cells. The expression of several other bcl-2 family members (bcl-X, ich-1, bax, bag, and bak ) and retinoid receptors (RARalpha, RXRalpha, and RXRbeta) was not affected by treatment with RAR- and/or RXR-activating retinoids; RARbeta RNA was undetectable before and after retinoid treatment. Thus, our cell model provides a useful tool in determining the genetic events mediating apoptosis as a response to RA, unobscured by events implicated in differentiation.