To assess more filly the metabolic fate and in vivo kinetics of dietary folate, we conducted an in depth study that followed the metabolism and excretion of radiolabeled folate from a single administration, through its many forms in the organs and tissues offolate-adequate rats. Twenty-two rats were equilibrated with an amino acid diet containing 1 mg folic acid/kg diet, then given 185 kBq ['H]f 1 o ic acid intragastrically. The isotopic label was followed through the tissues and in urine and feces for 32 days, every 8 hours for the first 48 hours. Individual folates, as their monoglutamyl forms, were separated and measured by high performance liquid chromatography (HPLC), and the peaks counted for 'H. Liver and kidney folates exhibited labeling (-0.8 kBq/g for each) by 8 hours post-dose. These organs exhibited a peak of [3H]folate at 40 hours post-dose, then slowly lost label from 48 hours to 32 days, with the exception of a rise in renal radioactivity at 16 days. Heart and spleen exhibited low levels of labeling at 8 hours, a peak at 32 hours, then a gradual loss of label. Testes and muscle (hind leg) showed very low levels of labeling throughout the study. Whole blood showed labeling almost entirely associated with 5-methyl-tetrahydrofolate (THF). Urinary excretion of intact folate occurred mainly as 5-methyl-THF, although excretion of products offolate catabolism exceeded urinary excretion of intact folates. Much of the labeled dose (35%) was excreted into the urine as catabolites and intactfolates by 32 days post-dose, whereas 15% of the label was lost through the feces by 32 days post-dose. Unexpected difSerences were observed among the specific radioactivity values among tissues (liver < kidney << testes, spleen, whole blood < heart << muscle). These may indicate different rates of clearance of labeled folates from these tissues or else the presence of kinetically slow pools comprising a significant portion of total folate in certain tissues (especially liver and kidney). The results of this study will be used to develop a compartmental model to simulate folate metabolism in folate-adequate rats.