The immediate-early gene zif268 (egr-1, NGFI-A, krox-24) encodes a transcription factor that has been proposed to play a role in differentiation and neuronal plasticity. zif268 mRNA is undetectable in unstimulated PC12D cells, a subline of PC12 characterized by accelerated differentiation in the presence of nerve growth factor, but is rapidly and robustly induced following exposure to muscarinic acetylcholine receptor (mAChR) agonists. Although PC12D cells express mRNAs for both m1 and m4 mAChR subtypes, induction of zif268 mRNA by mAChR agonists is apparently mediated exclusively by the m1 subtype because the induction is completely blocked by Dendroaspis angusticeps m1 toxin. Pretreatment of the cells with the specific protein kinase C (PKC) inhibitor GF109203X partially inhibits mAChR-mediated induction of zif268 mRNA. The remaining induction is blocked by chelation of extracellular calcium with EGTA. EGTA prevents the influx of calcium without blocking mAChR-mediated release of calcium from internal stores. These data indicate that both PKC and the influx of extracellular calcium play a role in the induction of zif268 mRNA following activation of the m1 mAChR. Transient increases in intracellular calcium levels resulting from the release of calcium from internal stores alone are not sufficient to induce zif268 mRNA. Rather, induction requires a prolonged elevation of intracellular calcium levels, which is sustained by the influx of extracellular calcium.