To study the tissue-specificity of folate deficiency, male Sprague-Dawley rats were fed folate-replete or folate-deficient diets with and without sulfonamide for 16 wk, and then injected with [3H]folic acid (1.5 nmol/kg). Rats were killed after 24 h, and the blood, urine and various organs were prepared for analysis of endogenous and 3H-labeled folate. Endogenous folate levels decreased due to folate deficiency to the greatest extent in the urine and plasma, followed by liver, kidney and other tissues (spleen, testis, lung and intestine), but no decrease was noted in the brain. Of all tissues of folate-deficient rats, the brain showed the greatest increase in incorporation of 3H-label from folate relative to folate-replete rats, with the largest effect in rats that were most deficient in plasma folate. Incorporation of label was increased due to folate deficiency in a number of tissues, with an inverse correlation with the tissue folate concentration. In contrast, hepatic [3H]folate incorporation was lower in folate-deficient rats than in folate-replete rats, with a direct correlation between endogenous folate concentration and the incorporation of labeled folate. These results show that the brain and other organs adapt to the development of folate deficiency because of greater incorporation of folate from exogenous sources. The lower incorporation by the liver of folate-deficient rats may result from the greater incorporation by other tissues.