Sulfated steroid hormones are commonly considered to be biologically inactive metabolites, but may be reactivated by the steroid sulfatase into biologically active free steroids, thereby having regulatory function via nuclear androgen and estrogen receptors which are widespread in the testis. However, a prerequisite for this mode of action would be a carrier-mediated import of the hydrophilic steroid sulfate molecules into specific target cells in reproductive tissues such as the testis. In the present study we detected predominant expression of the Sodium-dependent Organic Anion Transporter (SOAT), the Organic Anion Transporting Polypeptide 6A1, and the Organic Solute Carrier Partner 1 in human testis biopsies. All of these showed significantly lower or even absent mRNA expression in severe disorders of spermatogenesis (arrest at the level of spermatocytes or spermatogonia, Sertoli cell only syndrome). Only SOAT was significantly lower expressed in biopsies showing hypospermatogenesis. By use of immunohistochemistry SOAT was localized to germ cells at various stages in human testis biopsies showing normal spermatogenesis. SOAT immunoreactivity was detected in zygotene primary spermatocytes of stage V, pachytene spermatocytes of all stages (I-V), secondary spermatocytes of stage VI, and round spermatids (step 1 and step 2) in stages I and II. Furthermore, SOAT transport function for steroid sulfates was analyzed with a novel liquid chromatography tandem mass spectrometry procedure capable of profiling steroid sulfate molecules from cell lysates. With this technique, the cellular inward-directed SOAT transport was verified for the established substrates dehydroepiandrosterone sulfate and estrone-3-sulfate. Additionally, β-estradiol-3-sulfate and androstenediol-3-sulfate were identified as novel SOAT substrates.