An assay of folic acid in human serum is described involving HPLC fractionation of deproteinized serum prior to Lactobacillus casei assay. High specific activity [3H]folic acid is added as internal standard to 1 ml serum prior to deproteinization with perchlorate and an aliquot is applied to a C18 reverse-phase HPLC column. The folic acid fraction was assayed by L. casei microtiter plate assay. Intra- and interassay variations (CV) were 5.5 and 7.5% respectively for sera (n = 10) spiked with folic acid. The percentage recovery for folic acid additions of 10 and 20 ng/liter were 105.2 and 103.7%, respectively. The lower limit of detection was 1 ng folic acid/ml serum. Folates other than folic acid and 5-methyltetrahydrofolate were not detected. The assay permitted serial measurements to be made of folates in serum following an oral dose.