Currently, the only successful method for separating X and Y chromosome-bearing spermatozoa is fluorescence-activated cell sorting. Although effective, this technique is of limited usefulness to the animal breeding industry as it cannot produce the large volumes of sexed spermatozoa needed for artificial insemination. An attractive alternative would be to identify an immunological marker confined to one sperm type and, therefore, significant scientific effort has been expended in examining antibodies that appear to recognize approximately 50% of spermatozoa in an ejaculate. However, no sex-specific antigens have yet been identified from spermatozoa. Using the opportunity afforded by the development of sperm separation by fluorescence-activated cell sorting, we have made a thorough search for differences between X and Y chromosome-bearing bull spermatozoa using both biochemical and immunological methods. Techniques for radiolabelling surface membrane proteins, in conjunction with SDS-PAGE, failed to show any differences between populations. Similarly, a wide range of monoclonal antibodies raised to ejaculated, cauda epididymidal and testicular spermatozoa failed to distinguish between the X and Y chromosome-bearing spermatozoa. Only after analysis by high resolution two-dimensional SDS-PAGE was an indication obtained that X-specific proteins occur. However, these proteins are not associated with the surface membrane and further work is necessary to confirm their association with the X chromosome and to characterize them more fully. Our inability to detect sex-specific differences in sperm surface antigenicity suggests that further work on this immunological approach to semen sexing is unlikely to be profitable.