In this study, we evaluated the effect of a series of peripheral-acting benzodiazepines (BZDs), alone and in combination with recombinant human leukocyte (IFN-alpha A), fibroblast (IFN-beta), or immune (IFN-gamma) interferon (IFN), on the growth of human melanoma cells. Specific peripheral-acting BZDs caused a marked suppression in the proliferation of human melanoma cells. The effect on melanoma cell growth required 72 h exposure to the peripheral-acting BZDs and was not observed if the compounds were removed by 48 h. The relative potency of antiproliferative activity of a series of peripheral-acting BZDs on human melanoma cell growth did not correlate with the reported ability of these agents to bind to peripheral sites on the cell membrane of Friend erythroleukemia cells (FELC), nor did they correlate with the ability of these agents to inhibit [3H]thymidine incorporation in FELC, induce differentiation in FELC, or inhibit neurite outgrowth in nerve growth factor-treated rat pheochromocytoma (PC12) cells. The growth of human melanoma cells was also inhibited by various recombinant human IFNs, with IFN-beta displaying greater antiproliferative activity than IFN-alpha A or IFN-gamma. When the peripheral-acting BZD Ro7-3351, which displays growth inhibitory properties when used alone, was combined with IFN, the antiproliferative activity of the combination was greater than either individual compound exerted independently. The combination of IFN-beta plus Ro7-3351 was more active in suppressing HO-1 melanoma cell growth than other IFN preparations in combination with this peripheral-type BZD. Even when combined with a peripheral-acting BZD, such as Ro5-4608, which displayed only marginal antiproliferative activity against human melanoma cells when applied alone, growth suppression of the combination of this peripheral-type BZD with all three types of IFNs was more than additive. These studies suggest that specific peripheral-acting BZDs, both alone and in combination with recombinant IFNs, display novel antiproliferative activity toward human melanoma cells which may involve a different genetic locus than previously observed in other model cell culture systems.