Background: Experimental studies including longitudinal nitrogen balance studies could provide insight into protein metabolism in pregnancy. Objective: Our aim was to determine the development of nitrogen balance during pregnancy compared with nitrogen balance before pregnancy in women consuming imposed constant diets. We also tracked changes in muscle mass and lean body mass by measuring urinary 3-methylhistidine (3-MeH) and urinary creatinine. Design: Nitrogen balance was determined over 8 d in 12 healthy Dutch women before pregnancy and at weeks 12, 23, and 34 of gestation. Complete daily diets were supplied during each balance period so that each subject's energy, protein, and macronutrient intakes were similar in amount and composition in all 4 balance periods. Results: Throughout pregnancy there was no significant change in loss of nitrogen in feces and therefore no change in protein digestibility. The amount of nitrogen excreted in urine in late pregnancy (11.0 ± 1.4 g/d) was significantly (P < 0.01) less than in early pregnancy (12.6 ± 1.3 g/d). Nitrogen retention increased toward term, even though energy balance became progressively negative. The difference between the first (-0.4 ± 1.7 g N/d) and third (1.2 ± 1.6 g N/d) trimester was significant (P < 0.05). No differences were found in either 3-MeH or creatinine excretion between trimesters. Conclusions: These urinary nitrogen excretion and nitrogen retention data show that when the dietary supply remains constant, nitrogen balance increases toward the end of pregnancy, suggesting a more efficient use of dietary protein later in pregnancy. Urinary 3-MeH and creatinine excretion indicated no change in protein metabolism.