Astrocytes clearly play a role in neuronal development. An indirect mechanism of thyroid hormone (T3) in the regulation of neuronal development mediated by astrocytes has been proposed. T3 alters the production and organization of the extracellular matrix (ECM) proteins and proteoglycans, producing a high-quality substrate for neuronal differentiation. The present study investigated the effect of hypothyroidism on the astrocyte production of fibronectin (FN) and laminin (LN) as well as their involvement in neuronal growth and neuritogenesis. Our results demonstrated that the amount of both FN and LN were significantly reduced in cultures of hypothyroid astrocytes from rat cerebellum compared with normal cells. This effect was accompanied by reduced numbers of neurons and neuritogenesis. Similarly, the proportions of neurons and neurons with neurites were reduced in cultures on ECM prepared from hypothyroid astrocytes in comparison with normal cells. The proportion of both normal and hypothyroid neurons is strongly reduced in astrocyte ECM compared with cocultures on astrocyte monolayers, suggesting that extracellular factors other than ECM proteins are involved in this process. Moreover, treatment of hypothyroid astrocytic cultures with T3 restored the area of both FN and LN immunostaining to normal levels and partially reestablished neuronal survival and neuritogenesis. Taken together, our results demonstrated that hypothyroidism involves impairment of the astrocytic microenvironment and affects the production of ECM proteins. Thus, hypothyroidism is implicated in impaired neuronal development.