Secretagogin is a recently cloned human beta cell-expressed EF-hand calcium-binding protein. Converging evidence indicates that it exerts calcium sensor activity and that it is involved in the regulation of insulin synthesis and secretion. To obtain a potent tool for the extension of its functional analysis in rat in vitro systems, we cloned the rat homologue of human secretagogin. Using comparative sequence analysis, immunostaining, and immunoblotting, we demonstrated a high degree of sequence homology and similar tissue expression patterns of human and rat secretagogin. Highest rat secretagogin expression levels were found in pancreatic beta cells. Basing on newly generated anti-rat secretagogin antibodies, we established a rat secretagogin-specific sandwich capture ELISA and demonstrated release of secretagogin from viable Rin-5F cells. Interestingly, dexamethasone treatment of Rin-5F cells resulted in an increased secretagogin release rate, which was inversely correlated with the secretion of insulin. In contrast, the secretagogin transcription rate was markedly reduced. This resulted in a decreased intracellular secretagogin content under the influence of dexamethasone. Sucrose gradient cell fractionation analysis of Rin-5F cells confirmed the predominant cytosolic localization of secretagogin, with only limited association of secretagogin with insulin granules. Notably, the loss of intracellular secretagogin following dexamethasone treatment affected predominantly the insulin-granule associated secretagogin fractions. In conclusion, the sequence homology and the comparable tissue expression patterns of human and rat secretagogin indicate conserved intracellular functions. The effects of dexamethasone on the total Rin-5F secretagogin content and on its intracellular distribution might result in an impaired calcium sensitivity of dexamethasone treated insulin-secreting cells.