A cDNA fragment hybridizing with a transcript abundant in the periovulatory ovary was obtained while performing subtractive cloning on brook trout ovulatory and postovulatory ovarian tissue. Using this fragment as a probe, a 478 bp full-length cDNA was obtained by screening an ovulatory ovarian cDNA library. This cDNA presumably codes for an 88 amino acid protein that is structurally related to a new family of cysteine protease inhibitors characterized by the presence of a type I thyroglobulin motif in the amino acid sequence. Therefore, the protein was tentatively named an oocyte cysteine protease inhibitor (OCPI). On Northern blots, the OCPI cDNA hybridizes with a 0.5 kb transcript present in the ovary during the periovulatory period. The OCPI transcript and protein were localized to the cytoplasm of pre- and early-vitellogenic oocytes. On Northern blots of RNA from other tissues, the OCPI transcript was detected only in the ovary. On Western blots, OCPI was detected in the ovarian tissue at all periovulatory stages tested. The specific localization of both OCPI transcript and protein to pre- and early-vitellogenic oocytes and the structural similarity to protease inhibitors, suggest that OCPI might be involved in the protection of oocytes during the periovulatory period or in the regulation of yolk formation and degradation.