We examined cell surface molecules on spermatozoa of rainbow troutOncorhynchus mykissand their requirement during fertilization. A library of monoclonal antibodies was produced against surface antigens of rainbow trout sperm. Three types of antibodies were identified: most of the antibodies cross-reacted with two or more of the somatic cells tested; two cross-reacted only with brain tissue; and two – FS43 and FS 101 – were specific for sperm. All cross-reacted with sperm from chum salmonO. keta, coho salmonO. kisutch, and chinook salmonO. tshawytscha. The amount of sperm-specific antibody required to saturate the antigenic sites was significantly different for sperm from different males and was not related to total cell number. Fertility was reduced in a dose-dependent manner only for sperm preincubated with varying concentrations of antibody FS 101. Using indirect immunofluorescence, we observed that the two antibodies specific for spermatozoa restricted their binding to the head region of the sperm and were similar in their distributional patterns. These results support the hypothesis that a specific site present on the surface of rainbow trout spermatozoa participates in interactions necessary for fertilization.