The glandular or tissue kallikreins are a multigene family of serine proteases, of which 13 genes (rKLK1-13) have been identified in the rat and are expressed in a wide variety of tissues. Kallikrein-like enzyme activity has been detected during the periovulatory period in the gonadotropin-primed immature female rat ovary and suggested to play a role in the inflammatory-like response at ovulation. In this study, we examined whether this enzyme activity was due to local expression of a rat KLK gene family member. Ovarian RNA, prepared from gonadotropin-treated animals, was assessed for rKLK gene expression by reverse transcriptase-polymerase chain reaction (RT-PCR) with universal rKLK primers derived from highly conserved regions in the rat KLK genes. Southern blot analysis of the RT-PCR products, using oligonucleotide probes specific for the individual genes, indicated that five rKLK gene family members, rKLK1 (encoding true kallikrein), rKLK3, rKLK7, rKLK8, and rKLK9, were expressed at varying levels in the ovaries of both untreated control and gonadotropin-treated immature female rats. The identities of these five rKLK messenger RNAs were further confirmed by DNA sequence analysis of the PCR products. In situ hybridization of gonadotropin-treated ovaries localized rKLK3 and rKLK7 gene expression to the luteinizing granulosa cells of periovulatory follicles. In an enriched population of nonluteinizing granulosa cells prepared from estrogen-primed animals, we also demonstrated rKLK3, rKLK7, rKLK9, and rKLK12 (but not rKLK1 or rKLK8) expression, whereas all six genes were expressed in the ovaries of these animals. In summary, we have reported the expression of six KLK gene family members in the rat ovary and localized this expression primarily to the granulosa cell. The potential roles of these enzymes in ovulation or other aspects of ovarian, particularly granulosa cell, function are yet to be elucidated.