Milk and whey samples from healthy and inflamed udder quarters of 10 Ayrshire cows were analyzed for proteolytic activity using radial caseolysis procedures, a fluorogenic coumaryl peptide substrate, and casein agarose zymography. Free lysosomal enzyme activity (N–acetyl–beta–D–glucosaminidase) was used as the criterion for inflammation. All mastitic milk samples had proteolytic activity, tentatively identified as plasmin (comigration at Mr 83 000 and characteristic fragmentation). The plasmin activities in mastitic milk were on average 2.9 μg/ml (range 0.5–12.5) as measured by radial caseolysis. Milk or whey specimens from healthy quarters were all negative except 1 in which an activity of 0.1 μg/ml was found in both specimens. The caseolytic activities were totally inhibited by 50 KITJ/ml of aprotinin, a serine proteinase inhibitor from bovine lung. No free plasminogen activator (PA) activity was found in any of the samples. Howewer, according to zymographic analyses PA molecules corresponding to urokinase were found in healthy and especially in mastitic specimens. Analysis of plasmin may provide an alternative means of screening for mastitic milk samples.