Cyclic AMP response element binding protein (CREB) functions as an activity-dependent transcription factor in the nervous system. Increases in intracellular Ca2+ due to neuronal activity lead to the phosphorylation and subsequent activation of CREB. Although phosphorylation of CREB at Ser-133 is necessary for the stimulation of transcriptional activity, it is not sufficient. Here we demonstrate that in mouse cortical neurons, inhibition of the Ca2+-dependent protein phosphatase calcineurin by FK506 or cyclosporine A blocks CREB-dependent gene expression induced by depolarization without inhibiting depolarization-induced Ca2+ influx or CREB Ser-133 phosphorylation. Over-expression of a constitutively-active allele of the transducer of regulated CREB activity could not bypass the requirement for calcineurin activity. Stimulation of a CRE-luciferase reporter gene by depolarization was sensitive to FK506 throughout the entire time course of the transcriptional response, revealing that calcineurin activity is required to maintain CREB-dependent transcription. Stimulation of CRE-luciferase expression by forskolin and 8-Br-cAMP also required calcineurin activity. These results suggest that calcineurin functions as a critical determinant in shaping genome responses to CREB activation in cortical neurons.