Abstract The results of our previous studies have suggested that serum-induced inhibition of proximal tubular fluid absorption is due to complement-induced lysis of the tubular cells. The present study provides further evidence in support of this idea as well as other information pertinent to the mechanism of complement activation in vivo. 1. 1. The electrical resistance of the luminal brush border membrane is reduced drastically concomitantly with a drop in cell potential difference when serum is perfused intraluminally. 2. 2. Human C1 inhibitor (30–50 units/ml) does not significantly affect the inhibitory activity of human serum on fluid absorption, suggesting the non-involvement of the classical pathway. 3. 3. Reactive lysis reagents (C 5 6, C7, C8 + C9) partially inhibit fluid absorption when infused into the lumen. 4. 4. In contrast to our previous report (Sato, K. and Ullrich, K.J. (1974) Biochim. Biophys. Acta 354, 182–187), very fresh serum, 10-times diluted, can inhibit fluid absorption if perfused for 10 min. 5. 5. Both mouse and guinea pig serum, which are normally inactive, are activated to attack the tubular cells if 1/100 volume rat or rabbit serum is added to them. No such activation occurs by mixing guinea pig serum and mouse serum. The available data suggest that the presence of the later complement components but not the heat-labile factor (Factor B) or C3PA or C1 in the added serum is a prerequisite for mouse and guinea pig sera to be activated to inhibit fluid absorption.