These experiments were performed to determine why rabbit complement lyses tumor cells very efficiently, while not having particularly strong activity in hemolytic assays or in any other complement assay. The target cells used were human tumor cells coated with three different mouse IgG(2a) monoclonal antibodies, and complement from 5 mammalian species were tested. In antibody titration experiments, rabbit complement was found to lyse target cells at a relatively low antibody concentration, insufficient to allow lysis by complement of other species. Since this result was still observed after absorption of rabbit serum with target cells, the potency of rabbit complement cannot be attributed to the presence of natural antibodies. We then assayed C3 deposition on target cells, using two types of (125)I-labeled anti-C3 Abs to measure C3 deposition: goat antibodies specific for C3 of the human, guinea pig, rabbit, rat or mouse, and chicken antibodies to human C3 which cross-react with C3 of other mammals. Unexpectedly, complement of the human, rat, guinea pig, and BUB mouse deposited large amounts of C3 on the surface of target cells, while rabbit complement deposited 100-1,000 fold less. We discuss the possible reasons that C3 deposition does not correlate with cytotoxicity, and may indeed be inversely related. These data indicate that there is a fundamental difference in the complement cascade between rabbits and the other species tested. The potent lytic activity of rabbit complement is likely to be related to this difference, although the mechanism is not yet understood.