IGF-I is known to support growth and to prevent apoptosis in neuronal cells. Activation of the nuclear transcription factor cAMP response element-binding protein (CREB) has emerged as a central determinant in neuronal functions. In the present investigation, we examined the IGF-I-mediated phosphorylation and transcriptional activation of CREB in rat pheochromocytoma (PC12) cells, a cellular model for neuronal differentiation, and defined three distinct postreceptor signaling pathways important for this effect including the p38 mitogen-activated protein kinase (MAPK) pathway. CREB phosphorylation at serine 133 and its transcriptional activation as measured by a CREB-specific Gal4-CREB reporter and the neuroendocrine-specific gene chromogranin A was induced 2-3.3-fold by insulin-like growth factor (IGF)-I. This activation was significantly blocked (p < 0.001) by the dominant negative K-CREB or by mutation of the CRE site. IGF-I stimulated chromogranin A gene expression by Northern blot analysis 3.7-fold. Inhibition of MAPK kinase with PD98059, PI 3-kinase with wortmannin, and p38 MAPK with SB203580 blocked IGF-I-mediated phosphorylation and transcriptional activation of CREB by 30-50% (p < 0.001). Constitutively active and dominant negative regulators of the Ras and PI 3-kinase pathways confirmed the contribution of these pathways for CREB regulation by IGF-I. Cotransfection of PC12 cells with p38beta and constitutively active MAPK kinase 6 resulted in enhanced basal as well as IGF-I-stimulated chromogranin A promoter. IGF-I activated p38 MAPK, which was blocked by the inhibitor SB203580. This is the first description of a p38 MAPK-mediated nuclear signaling pathway for IGF-I leading to CREB-dependent neuronal specific gene expression.