This study investigated whether sauvagine and urotensin I change [Ca²⁺]_i in human epidermoid A-431 cells and whether these changes are correlated with their anti-edema properties in vivo. A-431 cells were used because they possess the corticotropin-releasing factor (CRF) receptor 2. Treatment with either sauvagine or urotensin I led to to an immediate increase in [Ca²⁺]_i, the magnitude of which depended on the concentration of the drug. Sauvagine was more effective than urotensin I, with a median effective concentration (EC₅₀) of 1.4 ± 0.2 fM, compared to an EC₅₀ of 66 ± 7 fM for urotensin I. Both were more effective at stimulating increases in [Ca²⁺]_i than CRF (EC₅₀ of 6.8 ± 0.1 pM). There was a correlation between the EC₅₀ for increasing [Ca²⁺]_i and the median effective dose (ED₅₀) for inhibiting edema induced by heating rat paw (r = 0.99). Removal of extracellular Ca²⁺ or incubation with La³⁺ eliminated the increase in [Ca²⁺]_i stimulated by either peptide. Pretreatment with a CRF receptor antagonist reduced the increase in [Ca²⁺]_i by these peptides. This occurred in an antagonist concentration-dependent manner, with median inhibitory concentrations (IC₅₀) of 1.99 ± 0.04 nM and 0.85 ± 0.04 nM, respectively. Both pertussis toxin (an inhibitor of G proteins) and U-73122 (an inhibitor for inositol trisphosphate (InsP₃) production) partially inhibited the increases. InsP₃ was measured to determine whether these peptides mobilized Ca²⁺ from an InsP₃-sensitive store. Both sauvagine and urotensin I increased InsP₃. The InsP₃ increases were inhibited by U-73122 and CRF antagonist, but not by removal of external Ca²⁺. Both peptides elevated protein tyrosine phosphorylation. In summary, these peptides increase [Ca²⁺]_i as a result of Ca²⁺ influx via CRF receptor-operated Ca²⁺ channels coupled to pertussis toxin-sensitive G proteins and a Ca²⁺ mobilization from InsP₃-sensitive Ca²⁺ pools. Their in vivo effectiveness at inhibiting edema is related to their respective capacities to stimulate elevations of [Ca²⁺]_i, supporting a role for intracellular Ca²⁺ in this process.