The second messenger pathways linking receptor activation at the membrane to changes in the nucleus are just beginning to be unraveled in neurons. The work presented here attempts to identify in striatal neurons the pathways that mediate cAMP response element–binding protein (CREB) phosphorylation and gene expression in response to NMDA receptor activation. We investigated the phosphorylation of the transcription factor CREB, the expression of the immediate early genec-fos, and the induction of a transfected reporter gene under the transcriptional control of CREB after stimulation of ionotropic glutamate receptors. We found that neither AMPA/kainate receptors nor NMDA receptors were able to stimulate independently a second messenger pathway that led to CREB phosphorylation orc-fos gene expression. Instead, we saw a consecutive pathway from AMPA/kainate receptors to NMDA receptors and from NMDA receptors to L-type Ca²⁺ channels. AMPA/kainate receptors were involved in relieving the Mg²⁺ block of NMDA receptors, and NMDA receptors triggered the opening of L-type Ca²⁺ channels. The second messenger pathway that activates CREB phosphorylation and c-fos gene expression is likely activated by Ca²⁺ entry through L-type Ca²⁺ channels. We conclude that in primary striatal neurons glutamate-mediated signal transduction is dependent on functional L-type Ca²⁺ channels.