To select a source of lymphocytes for the generation of an anti-sperm-biased combinatorial phage display library, venous blood was obtained from 34 vasovasostomy (vasectomy reversal) patients approximately 3 mo after surgery. Using a variety of immunoassays, serum was analyzed for antibodies against human spermatozoa, and a patient was selected on the basis of high titer of antibodies that recognized the equatorial region of the sperm head and inhibited sperm fertilizing capacity in vitro. Total RNA isolated from the stored lymphocytes of this individual was reversed transcribed, and γ1 (Fd) region and κ chains were amplified by polymerase chain reaction for the successful construction of an antibody phage display library. The library was panned against human spermatozoa to isolate sperm-specific phage that recognized the equatorial region of the sperm head. Three preparations of Fab were tested via the hamster egg penetration test. Each preparation significantly (p < 0.005) inhibited sperm-egg binding and fusion, with one preparation (designated Fab-G) causing complete inhibition. Sequence analysis of the κ light gene encoding Fab-G revealed a 93% homology with the light chain of human anti-human immunodeficiency virus gp120 p35 variable region. This technology may have a practical application in characterization of the immune response to spermatozoa and for the design of sperm-based contraceptive vaccines.