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Human milk pH was measured in 309 samples obtained from 52 women who had delivered at term and lactated for as long as 10 months thereafter. The mean pH decreased from 7.45 for colostrum to a nadir of 7.04 during the second week of lactation. Thereafter, the pH of milk remained between 7.0 and 7.1 until 3 months postpartum and then increased gradually to 7.4 by 10 months. The change in hydrogen ion concentration in milk was associated with corresponding changes throughout lactation in the concentration of citrate but not with the concentration of lactose. Lactose concentration increased gradually for 3 weeks; the concentration of saturated medium-chain fatty acids increased more rapidly. One interpretation of these findings is that the hydrogen ions and citrate generated by mammary secretory cell metabolism are used after the second week of lactation for de novo synthesis of fatty acids more rapidly than they are synthesized. Milk samples from ruminants were found to have concentrations of hydrogen ions and citrate that are greater than and pH that is less than the respective measurements in human milk. The significance for the recipient infant of the predictable changes in human milk pH during lactation and of the higher pH of human milk throughout lactation relative to bovine milk is unknown. However, drug excretion into milk, milk enzyme activity, milk leukocyte function, and neonatal gastrointestinal function are affected by ambient pH and may be influenced by the pH of milk.