Protease inhibitors (inhibiting trypsin, chymotrypsin, and elastase) were demonstrated in human milk from birth to 4 months after delivery. No pepsin inhibitor was found. The protease inhibitors were localized in the alpha 1-region--inhibiting trypsin, chymotrypsin, and elastase--and in a more cathodal region--inhibiting chymotrypsin--in agarose gel electrophoresis of human milk. alpha 1-antitrypsin and antichymotrypsin were demonstrated by crossed immunoelectrophoresis. Electroimmunoassay showed the concentration of alpha 1-antitrypsin in 1st day milk to be 10.9% and the concentration of antichymotrypsin was 116% of that of adult serum. The concentrations decreased during the 1st wk; from 1 wk to 4 months they were 1.6% for alpha 1-antitrypsin and 3.8% for antichymotrypsin of those of adult serum. One ml of human milk from the first 3 days inhibits, by enzymatic methods, 0-150 microgram (mean value, 70 microgram) trypsin. Milk from 1 week after delivery and later inhibits 0-65 microgram (mean value, 38 microgram) trypsin per ml.