SummaryProtease inhibitors and protease (caseinolytic, elastinolytic and esterolytic) activity were analysed in 190 milk samples from 94 mothers from day 1 to day 160 after delivery The main protease inhibitors in human milk are α1-antichymotrypsin and α1-antitrypsin. As measured by electroimmunoassay, the level of α1-antichymotrypsin in day 1 colostrum was higher than that in normal serum. Trace amounts of inter-α-trypsin inhibitor, α2-antiplasmin, α2-macroglobulin, antithrombin III, or antileukoprotease could be demonstrated. According to their protease inhibiting activity, the 53 milk samples from day 1–3 could be divided into two groups. (1) Presence of protease inhibiting activity (n= 35). Both α1-antitrypsin and α1-antichymotrypsin appeared intact and were able to form complexes with added trypsin or chymotrypsin although the major part of α1-antichymotrypsin showed a retarded electrophoretic mobility. The proteolytic activity was undetectable or low in these samples. (2) No protease inhibiting activity, in spite of the presence of immunoreactive inhibitors (n= 18). α1-Antichymotrypsin had a precipitate pattern similar to group 1, whereas α1-antitrypsin had a major fraction with slightly retarded mobility and two minor peaks in the α-1and β-regions. These precipitate patterns were unchanged on addition of human trypsin or chymotrypsin compatible with the presence of nonreactive inhibitor only. These samples had a caseinolytic and esterolytic activity with an electrophoretic mobility in the β-region.All samples from day 4 and later had a demonstrable protease inhibiting activity.