Human embryonic teratocarcinoma-derived Ntera2/cl.D1 (NT2) cells recapitulate many features of embryonic neuronal progenitor cells. Upon retinoic acid (RA) treatment they terminally differentiate into post-mitotic neuron-like cells (NT2-N), akin to human fetal neurons, thus representing an in vitro model of human neuron terminal differentiation. Experimental evidence also indicate NT2-N cultures as a potential source for cell transplantation therapy. The neurosteroids progesterone and its metabolite 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP) promote neurogenesis and show anti-neurodegenerative properties. This study's aim was to assess the neurosteroidogenic competence of NT2 cells during RA-induced neuronal differentiation. Radioimmunoassay measurements revealed progesterone only in NT2-N cultures (4 week RA). Accordingly, progesterone synthesis from (3)H-pregnenolone was absent in NT2 cells and increased during RA exposure, being highest in NT2-N. [(3)H]-pregnenolone metabolism, yielding [(3)H]-progesterone and [(3)H]-5alpha-dihydroprogesterone ([(3)H]-5alpha-DHP), was time-dependent and inhibited by trilostane, a 3beta-hydroxysteroid-dehydrogenase (3beta-HSD) inhibitor. Conversely, (3)H-progesterone metabolism, which yielded [(3)H]-5alpha-DHP > [(3)H]-3beta,5alpha-THP > [(3)H]-3alpha,5alpha-THP, occurred at all time points examined, though showing a nadir in cultures treated with RA for 1 and 2 weeks. The differentiation-dependent increase of progesterone accumulation matched 3beta-HSD type I mRNA expression and 3beta-HSD immunoreactivity, that co-localized with Map2a/b- and GAD67 in NT2-N. Hence, in vitro differentiated human neurons, while retaining progesterone metabolic activity, also become competent in progesterone synthesis. These findings suggest an autocrine/paracrine role of neuronal progesterone, either on its own or through its 5alpha-reduced metabolites, in fetal brain development and allow speculation that NT2-N-produced neurosteroids may contribute to the encouraging results of NT2-N transplants in animal models of neurodegenerative diseases.