Culture supernatants from monolayers prepared with guinea-pig peritoneal macrophages were found to contain functional D and C3 activity. Factor D was detected by consumption of C3 in the presence of culture supernatant, factor B and insoluble C3b. Preincubation of culture supernatant with anti-D IgG totally inhibited C3 consumption in the D assay which identified factor D as the B activating enzyme. The synthesis of D and C3 by macrophages was proven by the fact that cycloheximide in the culture medium strongly reduced the amount of detectable D and C3 and also incorporation experiments with 14C-labelled aminoacids resulted in the production by macrophages of radio-labelled D and C3. In addition radiolabelled B and P were also detected. The majority of the B protein appeared in its cleaved (Bb) form and, therefore, factor B escaped detection in the functional assay. For unknown reasons functional activity of P was not detectable. The enzyme responsible for B cleavage in the culture supernatants was identified as factor D. Activation of B by the D enzyme in culture supernatants probably occurred through the C3b-dependent feed back cycle of the alternative pathway. This is concluded from our observation that C3 was consumed on incubation of the culture supernatant at 37°, although at a low rate.