Like man, mouse has evolved a unique set of regulatory proteins which provide protection from complement-mediated damage to self membranes. The recently described mouse protein Crry/p65 has been shown to inhibit classical complement pathway C3 deposition on cell membranes in which it is expressed. In two distinct experimental systems, we now further delineate the regulatory activity of Crry/p65 and demonstrate its inhibitory effect on alternative complement pathway C3 activation. First, significant inhibition of mouse alternative pathway C3 deposition was demonstrated on neuraminidase-treated human K562 cells expressing recombinant Crry/p65. Second, using a baculovirus technique, recombinant Crry/p65 was synthesized as a soluble molecule and then purified. This molecule was found to inhibit mouse C3 deposition on the surface of zymosan, a potent alternative complement pathway activator. These studies, combined with our earlier findings, demonstrate that Crry/p65 can regulate both the classical and alternative complement pathways. Crry/p65 must, therefore, exert its effects prior to, or at the level of, the C3 convertases, in a fashion similar to that of human membrane cofactor protein and/or decay-accelerating factor. These studies provide further proof of the hypothesis that Crry/p65 is an evolutionarily unique, complement regulatory protein which has developed in mouse.