Recent evidence indicates that the N-terminal extracellular domain of receptors in the secretin-glucagon receptor family is responsible for ligand recognition. In this report, the N-terminal ectodomain of the human secretin receptor (HSR) was expressed in Escherichia coli, and the ability of this recombinant protein to interact with secretin was investigated by functional assays. The cDNA region encoding the N-terminal ectodomain of HSR linked to the polyhistidine fusion partner was expressed in E. coli. The resulting fusion protein was purified and used for competitive studies. A permanently transfected cell line with the HSR expressed was used in this study. The cell line was able to respond to secretin leading to the elevation of both intracellular cAMP and protein kinase-A activity. Using this cell line, incubation of secretin with the recombinant protein led to a dose-dependent inhibition of both cAMP production and protein kinase-A activity. These findings strongly suggested that the N-terminal ectodomain of HSR alone can act as a functional domain that provides a means to study ligand-receptor interactions of this receptor. The His-tagged recombinant HSR ectodomain may also be used for screening secretin-specific agonists and antagonists by affinity chromatography in the future.