Objectives were to sequence and examine the expression of the estrogen receptor β (ERβ) in the sheep ovary. The sequence of the ovine ERβ (oERβ) was determined using reverse-transcription polymerase chain reaction (RT-PCR) and cloning techniques. The reading frame of oERβ contained 527 amino acids and exhibited high overall homology with cow (98%), rat (88%), and human (88%) ERβ. In addition, an oERβ isoform having a 139-base pair deletion (oERβ1) was identified. The predicted amino acid sequence of this isoform is lacking the ligand-binding and carboxyl-terminal transactivation domains. The oERβ 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 oERβ protein was located in granulosa cells, the ovarian surface epithelium, endothelium, and Day 2 corpus luteum (CL). Weak immunostaining for ERβ was detected in the theca interna. Relative steady-state amounts of oERβ mRNA in the CL were determined using semiquantitative RT-PCR. Amounts of oERβ mRNA were greater (P < 0.05) during CL formation (Day 2) than at later stages. The oERβ to oERβ1 mRNA ratio was lower (P < 0.05) on Day 2 than on Day 10 or Day 30 due to a decrease in amounts of oERβ1. Results indicate that the oERβ 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 ERβ might regulate estrogen actions during early CL development in sheep.