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