The effects of a mild zinc-deficient state in humans were studied. Four male volunteers received restricted zinc intake for several weeks under strict metabolic conditions. As a result of dietary zinc restriction, a decrease in zinc concentration of plasma, erythrocytes, leukocytes, and urine was observed. Changes in the activities of zinc-dependent enzymes in the plasma such as alkaline phosphatase and ribonuclease were also related to the dietary zinc status. An adverse effect of zinc restriction on total protein, total collagen, ribonucleic acid, and the activity of deoxythymidine kinase (a zinc-dependent enzyme) in the sponge connective tissue of the two volunteers in whom this test was done was noted. During the zinc restriction period, the ammonia level in the plasma was elevated. Weight loss occurred in all subjects as a result of dietary zinc restriction. Inasmuch as the zinc-deficient state was mild, this study provides a basis for developing diagnostic criteria for zinc deficiency in humans.