Objectives were to sequence and examine the expression of the estrogen receptor beta (ERbeta) in the sheep ovary. The sequence of the ovine ERbeta (oERbeta) was determined using reverse-transcription polymerase chain reaction (RT-PCR) and cloning techniques. The reading frame of oERbeta contained 527 amino acids and exhibited high overall homology with cow (98%), rat (88%), and human (88%) ERbeta. In addition, an oERbeta isoform having a 139-base pair deletion (oERbeta1) was identified. The predicted amino acid sequence of this isoform is lacking the ligand-binding and carboxyl-terminal transactivation domains. The oERbeta protein and mRNA were determined in ovaries obtained from ewes on Days 0 (first day of estrus), 2, 6, and 10 of the estrous cycle and Day 30 of gestation. Immunohistochemistry showed that oERbeta protein was located in granulosa cells, the ovarian surface epithelium, endothelium, and Day 2 corpus luteum (CL). Weak immunostaining for ERbeta was detected in the theca interna. Relative steady-state amounts of oERbeta mRNA in the CL were determined using semiquantitative RT-PCR. Amounts of oERbeta mRNA were greater (P < 0.05) during CL formation (Day 2) than at later stages. The oERbeta to oERbeta1 mRNA ratio was lower (P < 0.05) on Day 2 than on Day 10 or Day 30 due to a decrease in amounts of oERbeta1. Results indicate that the oERbeta is a 527-amino acid protein expressed in specific cells of the ovary. Changes in relative amounts of full-length oERB and a deletion isoform in CL occurred during the estrous cycle, suggesting that these two types of ERbeta might regulate estrogen actions during early CL development in sheep.