Neurosteroids such as pregnenolone sulfate (PREGS) and dehydroepiandrosterone sulfate (DHEAS) are synthesized by specific cells within the brain, and are then delivered to their target sites in an autocrine or paracrine fashion. PREGS and DHEAS are also steroid hormone precursors in the peripheral circulation with important physiological functions. The present study tested the hypothesis that the heteromeric organic solute and steroid transporter, OSTα–OSTβ, mediates the transport of these compounds in steroidogenic cells. Measurement of [³H]DHEAS and [³H]PREGS transport in OSTα–OSTβ-expressing Xenopus laevis oocytes established that these neurosteroids are relatively high-affinity substrates, with apparent Km values of 1.5±0.4 and 6.9±2.1 µM, respectively. OSTα–OSTβ-mediated transport was cis-inhibited and trans-stimulated by a variety of steroid hormone conjugates, including DHEAS and PREGS, whereas the unconjugated parent compounds had no effect, suggesting that conjugated steroids are substrates for this transporter. Indirect immunofluorescence microscopy localized mouse Ostα and Ostβ and human OSTα and OST proteins to steroidogenic cells in the cerebellum and hippocampus, namely cells in the CA region of the hippocampus and in Purkinje cells. These observations were supported by analysis of Ostα and Ostβ mRNA levels in mouse Purkinje and hippocampal cells isolated via laser capture microdissection. In Ostα-deficient mice, serum DHEA and DHEAS levels were altered, as was the distribution of administered [³H]DHEAS, supporting a role of the transporter in steroid disposition. OSTα and OSTβ proteins were also located to the zona reticularis of human adrenal gland, the major region for DHEAS production in the periphery. Taken together, these results support the hypothesis that OSTα-OSTβ is a neurosteroid transporter, and suggest that it contributes to steroid hormone homeostasis.