The synthetic substrate carbobenzoxy-glycyl-prolyl-glycyl-glycyl-prolyl-alanine was used to estimate collagenolytlc activity in small sections of mammalian tissue and in aqueous extracts of homogenized tissues. A ninhydrln colorimetric analysis was used to determine the quantity of substrate digested. A variety of tissues taken from rabbits had the capacity to digest the substrate. Maximum activity by ovarian folllcular tissue occurred at pH 8.0. Treatment with para-chloromercuribenzoate or high temperature inhibited activity. Digestion increased linearly with the amount of tissue added to the incubated medium. Of nine commercial preparations of hydrolytlc enzymes, only bacterial collagenase digested the synthetic substrate. Metamorphosing tadpole tails had approximately 15 times the enzyme activity of nonmetamorphoslng controls. It is concluded that this substrate is useful in estimating nonspecific collagenolytic activity in small tissue sections.