We fed rats a defined amino acid, folate-free diet that did not contain sulfathiazole for 25 wk and examined the effects upon the content and distribution of folates in various tissues, based on the glutamic acid chain length and pteridine ring distribution. Compared with controls, rats fed the folate-deficient diet had lower folate concentrations in liver (11.10 +/- 1.04 vs. 27.11 +/- 2.72 nmol/g, P less than 0.001), kidney (4.79 +/- 0.65 vs. 11.69 +/- 1.40 nmol/g, P less than 0.01) and spleen (1.29 +/- 0.12 vs. 3.74 +/- 0.52 nmol/g, P less than 0.001), but not in brain (0.60 +/- 0.09 vs. 0.65 +/- 0.04 nmol/g, P greater than 0.1). The folate-deficient diet also resulted in changes in folate distribution reflected mostly by longer glutamate chains. In livers of the animals fed the folate-deficient diet the relative concentration of pentaglutamyl folates was lower and that of hexaglutamyl folates was higher, and there were also considerable amounts of hepta and octaglutamyl derivatives. Similar changes in folate distribution were observed in kidney and spleen. Brain folate distribution, however, was the same in the folate-deficient and folate-replete rats. The folate-deficient diet was without effect on the pteridine ring structure distribution in the various tissues except for small changes in the kidney. These results are discussed with respect to the relationship between tissue folate depletion and glutamic acid chain elongation and the possibility that folate conservation is different in the brain than in other tissues.