The follicular fluid of porcine ovaries contains an EDTA-sensitive enzyme activity for the synthetic substrate benzyloxycarbonyl-Val-Lys-Met-4-methylcoumaryl-7-amide. To investigate its characteristics and its identification, the enzyme was partially purified by ammonium sulfate fractionation followed by column chromatographies on DEAE-Cellulose and chelating Cellulofine columns. The enzyme activity was strongly inhibited by typical chelators, such as EDTA and o-phenanthroline, but after inhibition by EDTA the activity was completely restored with an appropriate amount of Zn 2+ and Co 2+ ions. It showed enzyme activity solely for benzyloxycarbonyl-Val-Lys-Met-4-methylcoumaryl-7-amide among the substrates tested. The molecular weight of the enzyme was estimated to be 400,000 by gel filtration. The enzyme activity in the fluid obtained from large follicles of porcine ovaries was significantly higher than that from smaller follicles. It appeared that the granulosa cell extract did not contain the metalloenzyme activity. Similar enzyme activities were detected in follicular fluids from bovine and human ovaries. These results suggest that the present enzyme is distinct from any other metalloendopeptidases thus far reported.