Four groups of weanling male rats were fed one of three iron-deficient diets (6, 18 and 23 mg iron/kg diet) or a normal iron-containing diet (41 mg iron/kg diet) for 30 d. The effects of the diets on various iron status parameters were determined and four enzymes were assayed: cytochrome P450 (P450) and NADPH cytochrome P450 reductase (P450-RED) in liver and intestine microsomes, and glucose-6-phosphate dehydrogenase (G6P-DH) and 6-phosphogluconate dehydrogenase (6PG-DH) in liver, intestine and erythrocyte cytosol. Rats fed 6 mg iron/kg diet were severely anemic, whereas rats fed 18 or 23 mg iron/kg diet were moderately or mildly iron-deficient, as shown by their hemoglobin levels, hematocrit, red blood cell parameters, erythrocyte protoporphyrin and liver iron stores. P450 concentration and P450-RED activity in liver were unaffected by iron deficiency, but P450 concentration was markedly lower in the intestine of the three iron-deficient groups than in the controls. Activities of G6P-DH and 6PG-DH were not impaired in liver or intestine, except that liver 6PG-DH activity of severely anemic rats was less than that of control rats. However, severe and moderate iron deprivation resulted in a stimulation of G6P-DH and 6PG-DH activities per million erythrocytes. These results demonstrate that even moderate iron deficiency may alter fundamental enzymatic systems intervening in drug metabolism and in the pentose phosphate pathway.