AbstractWe investigated mechanisms underlying nerve growth factor‐mediated morphological differentiation and expression of cholinergic neuronal phenotype. In PC12, but not PC12nnr5cells, nerve growth factor induces neurite‐like outgrowths and enhances cholinergic phenotype; stable expression of TrkA receptors in nnr5 cells (called B5P cells) restores morphological differentiation but not expression of choline acetyltransferase. Transfection with an AP‐1 luciferase reporter gene revealed that PC12 but not B5P cells expressed nerve growth factor‐induced functional AP‐1 activity. RT‐PCR analysis of nerve growth factor‐mediated changes in AP‐1 transcription factors showed rapid increases inc‐fosandjunBmRNA in PC12 and B5P cells, while increases inc‐junwere small. Using DNA–protein gel shift assays we determined that nerve growth factor stimulates AP‐1 binding in both PC12 and B5P cells, and identified c‐Fos, FosB, Fra‐1, Fra‐2, c‐Jun, JunB and JunD in AP‐1 complexes. In Fos/Jun functional luciferase reporter assays, nerve growth factor stimulated phosphorylation of c‐Fos in both PC12 and B5P cells, but phosphorylation of c‐Jun only in PC12, and not in B5P cells. These data indicate that mechanisms relating to AP‐1 transcription factor complexes underlying nerve growth factor‐mediated enhancement of cholinergic gene expression may differ from those required for morphological differentiation.