Peptide fragments of lactate dehydrogenase-C4 (LDH-C4) than contain antigenic sequences of the native protein were identified. This study describes the binding to murine and human spermatozoa of antibodies that were produced against synthetic peptides containing 2 of these sequences. Rabbits were immunized with peptides designated MC5-15 and MC211-220, conjugated to diphtheria toxoid (DT). Antisera from these rabbits were tested for binding to washed mouse epididymal sperm or human ejaculated spermatozoa using a solid-phase radioimmunoassay. Antisera bind to mouse sperm in this system at dilutions of 1:64,000. When these antisera are 1st absorbed with the native LDH-C4 molecule, significant inhibition of binding of sperm results. Antisera to both DT-MC5-15 and DT-MC211-220 bind to human sperm with similar but weaker patterns than seen with mouse sperm. The immune response to synthetic peptides containing antigenic sequences of LDH-C4 evidently includes antibodies that specifically bind to this enzyme on the surface of sperm. In addition, there are shared antigenic sequences between mouse and human LDH-C4, including the MC5-15 and MC211-220 peptides.