A 2.4-kilobase (kb) clone (kallikrein trout #14; KT-14) was isolated from a brook trout ovulatory cDNA library. KT-14 contains an open reading frame (ORF) of 768 base pairs (bp), presumably encoding a protein of 255 amino acids. The KT-14 cDNA also contains a 711 -bp 5' untranslated region and a 793-bp region downstream of the ORF that includes a 66-bp sequence repeated 12 times. The amino acid sequence of the KT-14 ORF is 41% identical to that of porcine complement factor D and 33% identical to that of porcine pancreatic kallikrein. On Northern blots of ovarian tissue, KT-14 hybridized with four transcripts of 1.8, 2.4, 2.9, and 3.2 kb. While the 3.2- and 2.4-kb transcripts were present in the ovary prior to meiotic maturation, they were significantly up-regulated at ovulation and at 12 h postovulation, respectively. Antibodies constructed against the recombinant KT-14 protein recognized one 30-kDa immunogenic protein in ovarian tissue and fluid. This immunogenic protein was significantly elevated in the tissue by ovulation. Using a follicle weight loss bioassay, we provide indirect evidence that mammalian kallikrein and related serine proteases can stimulate brook trout follicle contraction. Thus, one possible function of the KT-14 protein may be the regulation of oocyte expulsion at ovulation.