The high-temperature requirement factor A (HtrA) family of serine proteases is evolutionarily conserved from bacteria to mammals. We have previously identified Htra3 in the mouse and human (HTRA3) and reported its expression in the ovary. In this study, we analyzed the rat Htra3 gene and determined its developmental regulation in the rat ovary. We localized the rat Htra3 gene on chromosome 14q21 and identified two alternatively spliced mRNA variants. The two protein sequences deduced from these mRNAs enabled the prediction of the domain organization of the two protein isoforms. Our comparative analysis has established that the key gene features of Htra3 including its genomic structure, intron-exon junction and alternative splicing are well conserved among the mouse, rat and human. The similarities are even higher at the levels of primary protein sequence and protein domain architecture, suggesting that the functions of Htra3 are highly conserved during evolution from rodents to primates. We demonstrate that Htra3 expression in the rat ovary is developmentally regulated; expression was initiated on day 12 after birth and up-regulated during ovarian maturation with the highest levels found in the mature cycling ovary. In the mature ovary, Htra3 was expressed in granulosa cells, in a follicle-stage specific manner, with the level of expression being dependent on the positioning of the granulosa cells relative to the oocyte in late stage follicles. The luteinizing granulosa cells of the corpus luteum expressed the highest levels of Htra3. Collectively, these results suggest an important role for Htra3 in ovarian development, granulosa cell differentiation and luteinization.