In dairy cattle breeding, herd reproductive management is the primary focus, affecting a large part of the general costs. A negative association was observed between the level of milk production and fertility. Some studies have shown that a significant percentage of reproductive failure is attributable to semen quality; therefore, if reproduction management is based on artificial insemination, then it is important to assess the fertility level of the sires. In this study, proteomic analysis was used to compare the protein expression profiles from sperm of high- and low-fertility bulls. Comparative proteomic analysis showed that expression of several proteins [nine different two-dimensional electrophoresis (2-DE) spots] is related to fertility level (p <or= 0.05). These proteins are involved in sperm-egg interactions and cell cycle regulation. Differences in protein expression levels might explain reductions in fertility due to mistakes in sperm-oocyte communication or in cell cycle regulation. Proteomics of sperm can be a valuable tool to identify protein expression changes related to fertility; in particular, 2-DE-based proteome analysis is very useful for the characterization of spermatozoa protein expression related to high- and low-fertility rates. Furthermore, analysis of expression profiles could be critical to the identification of protein biomarkers of bull fertility.