CD46 (membrane cofactor protein) is a cell surface complement regulatory glycoprotein that facilitates enzymatic cleavage of complement component C3b; it is expressed by both human oocytes and acrosome-reacted spermatozoa. Murine anti-CD46 monoclonal antibody (mAb) has been reported to decrease significantly the ability of human spermatozoa to penetrate hamster oocytes. We have investigated the effect of purified anti-CD46 mAbs on spermatozoon-oocyte interaction in an autologous zona-free oocyte penetration test. Oocytes and/or spermatozoa were preincubated with either of two anti-CD46 murine mAbs, TRA.2.10 (a non-blocking mAb) and MH61 (a mAb that functionally blocks C3b-ligand binding activity), or a control isotype-matched mAb, in medium supplemented with human serum albumin. Preincubation of both spermatozoa and zona-free oocytes with TRA.2.10, but not MH61, caused a significant decrease in the number of oocytes showing sperm binding and pronuclear formation (9/23) compared with controls (21/26) in this complement component-depleted medium. This effect was not observed if oocytes or spermatozoa alone were preincubated. These data suggest that CD46 has a role in human spermatozoon-oocyte interaction at the level of the ooocyte plasma membrane, and indicate that a novel function other than direct C3b binding could be involved.