CRH induces corticotrope membrane depolarization and facilitates action potential firing. The increase in electrical excitability causes large oscillatory increases in cytosolic Ca2+ levels. In this study on highly enriched populations of cultured rat corticotropes, inhibitors were used to determine the contribution of the Na+ channel and Ca2+ channel subtypes to membrane excitability and cytosolic Ca2+ levels. Tetrodotoxin, an inhibitor of the voltage-dependent Na+ channel, inhibited a rapid initial component of the action potential, but generally did not influence spontaneous or CRH-induced firing frequency. Tetrodotoxin also had no effect on spontaneous or CRH-induced cytosolic Ca2+ levels. The L-type Ca2+ channel inhibitor nifedipine abolished spontaneous and CRH-induced action potentials and cytosolic Ca2+ transients, but did not eliminate the CRH-induced membrane depolarization or completely restore cytosolic Ca2+ to basal levels. Inhibition of P-type Ca2+ channels with omega-agatoxin-IVA decreased action potential firing frequency and reduced the CRH-induced increase in cytosolic Ca2+. The combination of nifedipine and omega-agatoxin-IVA abolished the CRH-induced rise in Ca2+, but did not abolish the membrane depolarization. Thus, cytosolic Ca2+ is mainly increased by CRH-induced action potentials that are completely dependent on L-type Ca2+ channels and partially regulated by P-type Ca2+ channels. CRH-induced Ca2+ entry also occurs independently of action potentials and is due to P-type, and possibly L-type, Ca2+ channels activated by the CRH-induced membrane depolarization.