Free-flow electrophoresis was used on bovine spermatozoa to test the hypothesis that there are surface charge differences between X and Y chromosome-bearing spermatozoa.Spermatozoa were deflected towards the anode by means of tromethamine (THAM) buffers of varying concentrations, and were separated into two populations under specific conditions. Experimental temperature and initial sperm motility had a significant effect on sperm distribution in the electric field. The results of DNA hybridization assays indicated an enrichment for Y chromosome-bearing spermatozoa.