The optimal conditions for performance of a sensitive functional assay for the human alternative complement pathway were studied. The serum dilution causing 50% lysis of rabbit erythrocytes in magnesium EGTA buffer is designated the APH50 titer. Optimal reaction conditions for the assay were pH 7.2, incubation temperature 37 degrees C, incubation time 60 minutes, and magnesium concentration 0.002 M. Lowering the ionic strength of the buffer from 0.150 M to 0.0125 M increased APH50 titers nearly 2.5-fold, but decreased the reproducibility of titers. Significant fluid-phase conversion of C3 at 37 degrees C in low ionic strength buffer was demonstrated by crossed immunoelectrophoresis. Using the optimal reaction conditions, in normal ionic strength buffer the mean APH50 +/- SD for 45 normal adults was 25.3 +/- 5.7 U/mL. Heparin, an inhibitor of the alternative pathway, decreased APH50 by 50% at a concentration of 100 U heparin/mL serum, and totally abolished alternative pathway hemolytic activity at 1,000 U heparin/mL serum, while lowering CH50 titers to a much lesser degree. When increasing doses of zymosan were used for complement activation in vitro, the per cent APH50 depletion at low doses of zymosan was at least twice the per cent depletion of CH50 or antigenic P, B, and C3. A striking dichotomy between nearly complete APH50 depletion and normal or near normal CH50 and hemolytic C4 levels was documented for a human burn patient and for a baboon infused with a lethal dose of Escherichia coli lipopolysaccharide. Therefore, we documented a substantially greater sensitivity of APH50 than of conventional complement determinations for detecting complement consumption by alternative pathway activators.