An increasingly exploited strategy for the isolation of stein cells is based on the increased efflux of Hoechst 33342 lipophilic dye mediated by ABCG2, all ATP-binding cassette transporter which is highly expressed it) various stern cells. We found ABCG2 expression to be present at later stages of spermatogenesis. Western Nor analysis using all anti-ABCG2 antibody revealed expression of a 72 kDa band in mature sperm obtained from mice, rats, bulls or humans. Immunocytochemistry Studies revealed acrosomal staining pattern of ABCG2 in spermatozoa. Experiments Using the Hoechst 33342 ABCG2 substrate and the ABCG2-specific inhibitor FTC demonstrated efflux activity of ABCG2 in mature sperm. Incubation of sperm in capacitating medium in the presence of the ABCG2-inhibitor FTC resulted in decreased cholesterol depletion compared to sperm incubated in the absence of FTC. Our results demonstrate that ABCG2 is expressed at the acrosome in mature sperm. ABCG2 may thus serve to mediate cholesterol removal. (c) 2008 Elsevier Inc. All rights reserved.