Thyroid hormone (T3) induces in vitro differentiation of astrocytes from the developing rat brain. T3 treatment induced the appearance of long processes in cultured cerebral hemisphere and mesencephalon astrocytes from embryonic and newborn rats. T3 treatment also produced a change in the morphology of cultured cerebellar astrocytes from 10-day-old rats, but not in cerebellar astrocytes from newborn rats. An increased expression of glial fibrillary acidic protein (GFAP) was also seen in the T3-treated newborn cerebral hemisphere and mesencephalic astrocytes. The morphological changes were induced earlier when the astrocytes were treated with conditioned medium (CM) obtained from cultures previously exposed to T3. Our results show that astrocytes from the developing rat brain are not homogeneous in their responsiveness to T3. Furthermore, the fact that CM produces a response similar to that obtained with T3 treatment but in less time, suggests that T3 might induce the secretion of factors by cultured astrocytes. These factors might, by an autocrine/paracrine effect, induce the expression of GFAP and differentiation in developing brain astrocytes.