Arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is the key enzyme in urea synthesis, hydrolyzing L-arginine into L-ornithine and urea. Arginase modulates levels of nitric oxide, creatine, and creatinine, likely by regulating intracellular L-arginine availability. The objective of the present study was to determine the arginase activity and concentration of urea and creatinine in colostrum and mature human milk obtained from nursing mothers. Our longitudinal biochemical analyses show that arginase activities and urea concentrations were the highest at the first day of lactation (colostrum). The decreasing enzyme activity and urea start at the second day, remaining at this level until the end of the first month of lactation (30th day). The concentration of creatinine in human colostrum and mature milk did not significantly change. The alteration of arginase activity between colostrum and mature milk may be a consequence of the transfer of arginase from the blood of the breast mother mammary glands into the colostrum and mature milk. The concentration of nutrients in colostrum and mature milk undergo alterations, probably to satisfy the requirements of the nursing infant for arginine, essential amino acids for human body growth, and normal physiology.