In cerebral hemisphere neuronal cultures derived from 15-day-old rat embryos, the addition of L-triiodothyronine (L-T3) or nerve growth factor (NGF) enhanced the expression of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in a dose-dependent manner. When cultures were supplemented with both agents at maximal effective concentrations, the stimulation in ChAT and AChE activities was significantly greater than the sum of the individual effects. Conversely, when the cultures were exposed to astrocyte conditioned medium grown in the presence or absence of L-T3 (CM + L-T3 or CM-L-T3). laminin and fibroblast growth factor (FGF), ChAT and AChE activities were not stimulated above those of control cultures when added alone or in combination with L-T3. Furthermore, L-T3, NGF, CMs, laminin and FGF did not affect AChE+ cell survival, but significantly increased neurite outgrowth and branching with NGF and L-T3 being the most powerful agents followed by CMs, laminin and FGF. Additionally, the simultaneous addition of L-T3 with either laminin or FGF in culture, caused an additive effect of L-T2 in the neurite density of AChE+ cells with both agents. This study shows that (1) thyroid hormones do not act through the regulation of soluble neurotrophic factors produced by astroglial cells, (2) thyroid hormones interact with the effect of NGF on ChAT and AChE activities, (3) the regulation of ChAT and AChE activities and the neurite outgrowth are independently regulated. and (4) the regulation of ChAT and AChE activities is very specific compared with that of neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)