Cocaine is a major drug of abuse among women of child bearing age nationwide. We showed in our previous studies that cocaine inhibits neuronal differentiation in a dose-dependent fashion in nerve growth factor(NGF)-stimulated PC12 cells, without affecting cell viability. Cocaine effects related to duration of exposure. Only partial recovery was noted upon cocaine removal. NGF exerts its biological effects through interaction with the receptor tyrosine kinase, which activates a cascade of intracellular signaling proteins, specific immediate early genes (IEG) including a transient peak in c-fos expression, and induction of late genes expression, leading to the neuronal phenotype. We hypothesized that cocaine alters the expression of c-fos and therefore blocks NGF-mediated neurite outgrowth. In this study we examined the time-course of cocaine's effects on the expression of c-fos in NGF-induced PC-12 cells, treated with NGF 20 ng/ml (the ED50) and cocaine 10 μg/ml (a moderately toxic dose). C-fos expression was determined at 0.5, 2, 4, 6, 24 and 72 hr after exposure to NGF with and without cocaine, by RT-PCR analysis. Total RNA was isolated from cells, and levels of c-fos mRNA were estimated using gene-specific primers. A multiplex RT-PCR system was used with cyclophilin as an internal standard. The levels of c-fos mRNA were quantitated and reported relative to cyclophilin (c-fos/cyclo). In both control and experimental conditions, c-fos/cyclo ratio was maximal at 0.5 hr. In control cells, c-fos expression declined rapidly to less than 5% of the 0.5hr value. However, in the cocaine treated cells, c-fos expression persisted through 72 hr, exceeding that in controls by 3-6 fold. Overexpression of c-fos inhibits NGF-induced PC12 differentiation probably through blocking NGF-mediated exit from the cell cycle, and by blocking the induction of late genes expression. Cocaine inhibitory effects on NGF-induced differentiation may be attributed to cocaine's striking effects on the temporal expression of c-fos. In rat forebrain cultures, cocaine induced c-fos expression through the activation of D-1 receptors. Further studies will be required to determine if blocking c-fos expression would reverse cocaine effects on differentiation, to examine the role of D-1 receptor activation in c-fos expression and to explore the effects of cocaine on other IEGs.