An estrus stage-specific proteinase of an estimated molecular weight of 600 kDa was detected in uterine luminal fluid of the rat. Estradiol administrated sc to adult female ovariectomized and immature rats induced its production. The 600 kDa protein was stable to sodium dodecyl sulfate polyacrylamide electrophoresis under reducing conditions, and retained its proteolytic activity. The proteolytic activity was not inhibited by the following proteinase inhibitors: 1,10-phenanthroline, leupeptin, phenylmethylsulfonyl fluoride and pepstatin A. The 600 kDa proteinase was not detected in the uterus or in the spent medium in which uteri obtained from estrus stage of normal cycling rats or from estradiol-treated adult ovariectomized rats were cultured. Its presence in the uterine fluid suggests possible involvement in the fertilization process, viability of zygotes and/or implantation of blastocysts.