A partial cDNA, corresponding to the mouse prostaglandin E2 receptor subtype EP1, was isolated from mouse brain cDNA using a degenerate primer PCR strategy. Using the cDNA fragment as a probe, the EP1 receptor gene was isolated and characterized. The gene consists of three exons, of which the first is non-coding, and is contained within a 3.5-kb region. The coding nucleotide sequence determined is identical to that of the published mouse EP1 cDNA. The positions of the introns correspond to those of the thromboxane A2 and prostaglandin D receptor genes. No alternative splicing of the EP1 receptor gene could be detected. PCR and specific primers designed from the genomic sequence were used to amplify the coding part of the isolated gene from kidney cDNA. The cDNA obtained was cloned into a eukaryotic expression vector, and stably transfected Chinese hamster ovary cell lines were established. The cells respond to prostaglandin E2 with intracellular Ca2+ mobilization, as expected for this prostanoid receptor subtype. In situ hybridization was used to localize the EP1 receptor transcript in different mouse tissues. Significant hybridization was detected only in the collecting ducts of the kidney, and in the paraventricular and supraoptic nuclei of the hypothalamus. The expression of the EP1 receptor in the hypothalamus suggests that this prostanoid receptor is involved in mediating the fever response evoked by prostaglandin E2.