As mastitis is associated with leakage of small molecular weight plasma proteins, such as α1-antitrypsin, into milk, this antitrypsin can be used as an indicator of mastitis. A colorimetric procedure was developed for large scale monitoring of milk antitrypsin activity, using microtitration plates and the Multiskan system. The effect of stage of lactation and age of the cow on the antitrypsin concentration and its interrelationship with other mastitis indicators (bovine serum albumin (BSA), somatic cell count) was analysed by computer programs on 1029 cows. Milk antitrypsin activity was high after parturition owing to colostral inhibitors. After the first month of lactation the assay measures only blood-derived antitrypsin and is a good indicator for detecting an increased permeability between blood and milk due to mastitis. Increasing lactation number only slightly affected the antitrypsin and BSA concentrations whereas somatic cell content was markedly affected.