Monolayer cultures from hypothalami of embryonic, fetal and postnatal rats were established to study the development of oxytocin-secreting neurons in vitro. Particular culture conditions, known to enhance the survival and growth of different types of neural cells in vitro, were used to investigate the conditions necessary for the appearance and survival of these peptide-producing cells in culture. They included increasing the concentration of potassium in the culture milieu and/or the addition of triiodothyronine (T(3) ). The use of immunocytochemical procedures with a monoclonal antibody that recognizes oxytocin-associated neurophysin (NP-OT) and polyclonal antibodies specific for oxytocin permitted us to identify the neurons. In cultures derived from embryonic (E16-E17) hypothalami, no NP-OT- or oxytocin-positive neurons were detected and their appearance could not be provoked by increasing extracellular potassium concentration or by administration of T(3) . On the other hand, in cultures obtained from fetal (E18-E19) rat hypothalami, a few neurons showed immunoreactivity for NP-OT (but not for oxytocin); the immunoreactivity was localized essentially in somata and proximal portions of neurites. When 10(8) M T(3) was included in the culture medium, there were cells showing immunoreactivity not only for NP-OT, but also for oxytocin, visible in somata and in dendritic- and axonal-like processes. In comparison, T(3) did not influence the total number of neurons developing in these cultures. Lastly, in cultures derived from young postnatal (PO-P2) rat hypothalami, NP-OT- and oxytocin-immunopositive neurons were found regularly; their appearance did not require any special pretreatment of the cultures. In all cultures, high extracellular potassium concentration (25 mM) resulted in a general improvement in the survival of neurons but did not induce the appearance of more oxytocin-immunoreactive cells. Our observations support in vivo results showing that although presumptive oxytocin-producing cells appear early in the development of the hypothalamus, their maturation, and in particular, their ability to produce oxytocin, occurs late at the time of birth. A factor that selectively enhances their differentiation, is the thyroid hormone, T(3) .