The cDNA for the full-length porcine estrogen receptor β (ERβ) and an alternatively spliced transcript with a deletion of exon 5 (ERβδ5) was cloned from pig ovary. RNase protection assays revealed that ERβ mRNA was expressed in the preovulatory follicles and early, midluteal, and regressing corpora lutea (CL) of eCG ± hCG-primed gilts. ERβ and ERβδ5 transcripts were shown by semiquantitative reverse transcription polymerase chain reaction to be expressed at a ratio of approximately 2:1 in granulosa cells, small, medium, and large antral follicles, and midluteal phase corpora lutea of unprimed animals. Immunoreactive ERβ proteins corresponding to the size of in vitro translated ERβ and ERβδ5 were detected by immunoblot. Full-length ERβ was detected in granulosa, small, medium, and large antral follicles, and midluteal phase CL of unprimed animals. Putative ERβδ5 immunoreactive bands were abundant only in granulosa cell extracts. In COS-1 cells, transfected ERβδ5 had no effect on basal transcription of an estrogen-responsive reporter construct but did repress wild-type ERβ transactivation when cotransfected at 10-fold excess plasmid. No repression of ERα transactivation was observed. In primary granulosa cell cultures, transfected ERβδ5 plasmid did not inhibit basal reporter activation. ERβδ5 was shown by immunofluorescence to localize to the nucleus in transfected COS-1 cells. In vitro translated ERβδ5 proteins bound estrogen response elements in DNA in electrophoretic mobility shift assays, as indicated by supershift analysis. ERβ is abundant in porcine ovary, and a naturally occurring splice variant missing exon 5 may have biological function.