Heat-killed kidney cells (HKKC) incubated with serum or purified C3 and factors B and D stain intensely for C3 by immunofluorescence. Binding of complement (C) did not occur if ethylenediaminetetraacetic acid (EDTA) was added to the C source or if factor B or D was omitted from the mixture of alternative pathway components. When the C-treated cells were washed, then incubated in purified C3 containing 10 mM EDTA, C3 conversion ensued, confirming the presence of a cell-bound C3 convertase. C3-reactive cells were not generated if EDTA was present during formation of the convertase. The addition of C3 nephritic factor (C3NeF) during formation of the C3 convertase from alternative pathway components greatly enhanced subsequent C3 cleavage, presumably by stabilizing labile C3bBb sites; however, binding of C3NeF did not appear to extend the half-life of the convertase, which was 35 min at 37 degrees C in each case. In contrast, little or no effect on C3 conversion could be detected after the addition of equivalent C3NeF to serum and HKKC. Thus, C activation by HKKC involves formation and activity of a cell-bound C3 convertase, which under certain circumstances can be stabilized by C3NeF.