The pineal gland (PG) is a source of several trophic factors. In this study, PG and superior cervical ganglia (SCG) from Sprague-Dawley neonates (1-day-old) were co-cultured to test the hypothesis that endogenous release of PG NGF (or an NGF-like cytokine) is sufficient to promote survival of SCG neurons. Neuronal density of SCG neurons was significantly enhanced when co-cultured with PG for 7 days compared to SCG cultured alone. SCG survival and neurite formation in PG co-cultures was less than in SCG treated with exogenous NGF (100 ng/ml). The neurotrophic effect of PG co-cultures was abolished when 1% anti-NGF was added to the medium. Co-cultures of SCG neurons with established 7-day PG cultures induced extensive SCG neurite formation within 24 hr compared to SCG co-cultured with 1-day PG cultures. This suggests that PG neurotrophic effects are due to PG non-neuronal cells (nnc) that proliferate to confluency by 7 days in culture. S-antigen-positive pinealocytes did not proliferate in culture. There was decreased SCG survival when neurons were seeded onto PG cultures that had been previously killed by drying, which suggests that the neurotrophic effects of nnc are not substrate-dependent. Immunocytochemical characterization of PG nnc revealed a heterogenous mixture of astrocytes, macrophage/microglia, and fibroblasts. These findings support the hypothesis that NGF is actively secreted by PG and that nnc are the principal source of this neurotophin.