Thrombin-like enzymatic activity was measured in mouse brain homogenates and slices by cleavage of a peptide substrate, N-p-Tosyl-Gly-Pro-Arg-7-amido-4-methylcoumarin. The activity was localized mainly to white matter. However, it was not affected by specific thrombin inhibitors, and was found to represent the sum of at least two enzyme activities, a prolyl endopeptidase and an aminopeptidase. By specifically inhibiting this endogenous activity in combination with exogenously added thrombin, mouse brain tissue was shown to express a capacity of thrombin inhibitory activity equivalent to 0.2 mU thrombin/mg brain tissue. The present study offers a simple and reliable method for measuring total thrombin inhibitory activity in brain.