The antitumor effect of human natural and recombinant interferon-gamma (IFN-gamma) was evaluated in human osteosarcomas grown as xenografts in nude mice. IFN-gamma was given as daily subcutaneous injections, alone or in combination with IFN-alpha. The growth of two out of three tested osteosarcomas was inhibited by 2 x 10(5) IU of natural IFN-gamma. A five times higher dose of recombinant IFN-gamma, as compared with natural (n) IFN-gamma, was needed to obtain growth inhibition of one osteosarcoma. This difference in dose-response could be explained by differences in pharmacokinetics. Hence, subcutaneously administered natural IFN-gamma gave 10 times higher serum levels than obtained with the recombinant type. Combination treatment with IFN-alpha and IFN-gamma induced a potentiation of the antitumor effect in one osteosarcoma. In another osteosarcoma, 2-4 x 10(5) IU of nIFN-gamma did not effect tumor growth and could not potentiate the antitumor effect of 2-4 x 10(5) IU of nIFN-alpha. By using DNA analysis in cell suspension and tissue section, the proportion of aneuploid tumor cells within the xenograft could be estimated. This analysis showed that the antitumor effects of IFN were more pronounced than mere measurement of tumor volume suggested. IFN-inhibited tumors were partly replaced by fibroblasts or bone tissue. In conclusion, at the doses given nIFN-gamma appeared to have similar antitumor effects as IFN-alpha in two osteosarcomas, whereas one was sensitive to only IFN-alpha. Combination IFN treatment induced a potentiation of the antitumor effect in one osteosarcoma but not in another. The differences between the osteosarcomas in obtained antitumor effect of IFN treatment probably reflects individual IFN sensitivity and demonstrates the importance of assessing several tumors of the same neoplastic entity.