The mechanism of increased antitumor activity when human lymphoblastoid interferon [HuIFN-alpha(Ly)] and the drugs cyclophosphamide and Adriamycin are used in combination on a human tumor xenograft in nude mice has been investigated. HuIFN-alpha(Ly) did not affect hepatic levels of the drug-metabolizing enzymes cytochrome P-450 or the glutathione S-transferases. In contrast, mouse interferon caused significant and differential changes in the isozymic forms of these enzymes. However, addition of mouse interferon to the HuIFN-alpha(Ly)/cyclophosphamide or Adriamycin combinations had no effect on the final result, and did not increase the toxicity of the combination therapy. These data provide evidence that the increased activity of the combination therapy is due to effects on the tumor rather than on the host. Further studies showed significant perturbations in the tumor cell cycle after in vivo combination therapy. Cyclophosphamide caused an accumulation in G2 and the addition of HuIFN-alpha(Ly), which alone caused little change in cycle distribution, delayed this G2 block and strongly increased the number of cells in S phase. A similar, although less pronounced, effect was seen with HuIFN-alpha(Ly)/Adriamycin therapy. The increase in S phase seen in combined therapy may account for the synergy seen.