Retinoids have been shown to be clinically useful in the biological therapy of certain myeloid and T-cell malignancies, whereas CD20 has proven to be an effective target in B-cell lymphoma immunotherapy. Both retinoic acid derivatives and anti-CD20 monoclonal antibodies have also been shown to induce apoptosis of malignant cells in vitro. Retinoid-induced apoptosis is thought to be mediated by nuclear retinoid receptor binding and transcriptional activation, whereas CD20 ligation appears to initiate transmembrane Ca(2+) influx with resultant programmed cell death. In this report, we evaluate the in vitro effects of N-(4-hydroxyphenyl) retinamide (4-HPR) with and without anti-CD20 antibodies in B-cell lymphoma lines. We demonstrate that 4-HPR inhibits the growth of malignant B-cells beyond that of all-trans-retinoic acid and 13-cis-retinoic acid. We also show that this 4-HPR-mediated growth inhibition is attributable to apoptosis, is consistent across a variety of malignant B-cell lines (Ramos, Ramos AW, SU-DHL4, and Raji), peaks at 96 to 144 h, and is attainable with concentrations as low as 2 microM. As with CD20-mediated apoptosis, we show that the final common pathway includes caspase activation that can be blocked by 2-val-Ala-Asp-fluoromethyl ketone (z-VAD), a specific inhibitor of caspase function. Coincubation of a 2 microM concentration of 4-HPR and the anti-CD20 antibodies rituximab and tositumomab exhibited a supra-additive increase in levels of apoptosis induction of 24% (P = 0.009) and 42% (P = 0.0019) relative to expected additive levels of these same agents. These in vitro findings suggest that the potential in vivo synergy of these well-tolerated drugs may augment the previously demonstrated clinical activity of anti-CD20 monoclonal antibodies in the treatment of B-cell malignancies.