Mammary amylase is a possible alternate pathway of digestion of glucose polymers and starches, that is most important in early infancy when pancreatic amylase is low or absent in duodenal fluid and responds poorly to stimuli. Human breast milk contains 1000-5000 units of amylase/liter. In order to evaluate the likelihood that a significant proportion of mammary amylase activity would withstand passage through the stomach, purified and unpurified mammary amylase were exposed to acid and pepsin in vitro to simulate the gastric environment found in young infants. Both purified and unpurified enzymes were stable at pH 7.5 with little or no activity lost after 4 h, and approximately 80% retained at 6 h. When incubated at pH 3.5, one-third of unpurified enzyme activity was retained for 6 h; in contrast, the purified enzyme was acid labile losing 80% by 2 h. Addition of bovine serum albumin or breast milk proteins to purified enzyme protected the activity. When unpurified enzyme was exposed to a stepwise decline in pH from 6.5 to 3.5 over 4 h, 50% of the original activity was retained. Unless the concentration was greater than or equal to 3750 units/ml, the addition of varying concentrations of pepsin to defatted breast milk incubated at pH 3.5 did not affect any greater decay of enzyme activity despite evidence of peptic digestion of proteins in the reaction mixture. This study supports the possibility that ingested mammary amylase could retain a significant proportion of its original activity after exposure to acid and pepsin in the stomach of young infants.