Interleukin-1alpha (IL-1alpha) has potent acute antitumor activity in vivo and can enhance the efficacy of chemotherapeutic drug-mediated antitumor responses. Studies were undertaken to examine the ability of IL-1alpha to enhance the activity of cyclophosphamide (CTX) administered in combination with carboplatin. To determine the in vivo effect of IL-1alpha, CTX and/or carboplatin, mice bearing 14-day RIF-1 tumors were treated on day 0 with a concurrent i.p. injection of varying doses of CTX (5-150 mg/kg), human IL-1alpha (125 microg/kg), and carboplatin (50 mg/kg) and examined 24 h later for the surviving fraction by the in vivo excision clonogenic-tumor-cell assay. Even at the lowest doses of CTX, IL-1alpha significantly enhanced the clonogenic tumor cell kill when compared to treatment with CTX alone. When carboplatin was added to the treatment schema, significantly greater clonogenic cell killing and tumor regrowth delay were observed as compared to any agent alone or a two-drug combination (CTX/IL-1alpha or CTX/carboplatin). Significant enhancement was observed even at low doses of CTX in combination with carboplatin and IL-1alpha. The interaction between the three-drug combination was found to be synergistic as determined by the median dose effect with significant dose reduction apparent for IL-1alpha and CTX when used in this combination. These results demonstrate that IL-1alpha can synergistically enhance the antitumor efficacy of CTX and the combination of CTX and carboplatin.