A growing number of estrogen receptor beta (ER beta) splice variants are reported. Several of these have been discovered in testis, but with few exceptions little is known about their cellular localization. The aim of this study was to identify and elucidate the mRNA expression pattern of the different ER beta splice variants in human testicular cells. Northern analysis was performed on whole testis and fractions enriched in germ cells from untreated men and from estrogen-treated men undergoing sex change surgery. Probes were constructed in order to systematically screen for and identify various ER beta splice variants. Several ER beta bands were observed in the human testis, in which splice variants constituted the major part of total ER beta transcripts. Interestingly, only two ER beta wild-type transcripts were detected. These seem to be virtually absent from the haploid germ cells and are probably mainly located in somatic cells and/or primary spermatocytes. Several novel ER beta deletion variants were found in high levels in the haploid germ cell fractions and were nearly absent in testicular cells from the estrogen-treated men. The cell-dependent distribution raises the question whether splice variants may have specific functions in spermatogenesis, and whether the differential splicing of ER beta is regulated in a cell-specific manner.