Abstract Corticotropin-releasing factor (CRF) stimulates adrenocorticotropin (ACTH) release via the adenylate cyclase/cAMP-dependent protein kinase system. Because calcium is necessary for receptor-mediated release of ACTH, we have examined the effect of CRF on 45Ca 2+ uptake in a corticotroph cell line model, AtT-20. Treatment of AtT-20 cells with CRF (10 −9 − 10 −6 M) resulted in dose- and time-dependent increases in 45Ca 2+ uptake, up to 2.2-fold above control values. The effect was statistically significant at 1 min and persisted for at least 10 min. Treatment with forskolin (1–30 μM), 8-Br-cAMP (0.5 mM), cholera toxin (CT, 100 ng/ml) and K + (20 mM) also increased cell-associated 45Ca 2+. The effect of K + was completely blocked by nifedipine (100 μM), whereas the effects of CRF (10 −8 M) were only partially inhibited by this calcium channel antagonist. These data suggested a role of voltage-dependent calcium channels in 45Ca 2+ uptake. Short term pretreatment (1–2 h) of AtT-20 cells with CRF (10 −8 M) significantly desensitized both CRF-stimulated cAMP accumulation and ACTH release, but did not attenuate CRF-stimulated 45Ca 2+ uptake. Pretreatment with CRF (10 −8 M) for 4 h did not alter CT- or forskolin-stimulated cAMP accumulation and ACTH release. This suggests that the molecular mechanisms of desensitization are proximal to adenylate cyclase. Conversely, long term pretreatment (24 h) of AtT-20 cells with CRF (10 −8 M) induced significant desensitization of CRF-stimulated 45Ca 2+ uptake. These results indicate that CRF stimulates calcium uptake in AtT-20 cells via cAMP-dependent and cAMP-independent mechanisms, and that the cellular mechanisms involved in desensitization of cAMP accumulation and ACTH release and those involved in desensitization of calcium uptake are qualitatively different.