The only known and measurable difference between X- and Y-chromosome bearing spermatozoa is the small difference in their DNA content. The X sperm in the human carry 2.8% more DNA than the Y sperm, while in domestic livestock this difference ranges from 3.0 to 4.2%. The only successful sperm separation method, flow cytometric sorting, is based on this difference in DNA content. Using this technique, X and Y sperm populations with purities greater than 90% can be obtained. The number of spermatozoa that can be sorted in a given time period, however, is too low for application of this technique in routine artificial insemination. Therefore, the search for a marker other than DNA to differentiate between X and Y sperm remains of interest in order to develop a method for large scale X and Y sperm separation. The aim of the present study was to investigate whether porcine X and Y sperm contain some difference in their plasma membrane proteins. The flow cytometric sorting of sperm enabled a direct comparison of the proteins of the X and Y sperm populations. High resolution two-dimensional (2-D) electrophoresis was used; however, adaptations were needed to enable its use for analysis of proteins of flow cytometrically sorted sperm, both in the sorting procedure, membrane protein solubilization, and in the 2-D electrophoresis. Up to 1000 protein spots per gel could be detected and quantified. Comparison of the 2-D protein patterns revealed differences in protein spots between sperm of two individual boars. However, no differences in protein spots between the X and Y sperm fractions were found. These results provide additional support for the view that X- and Y-chromosome bearing spermatozoa are phenotypically identical, and cast doubt on the likelihood that a surface marker can provide a base for X and Y sperm separation.