For the first time nonformylated tetrahydrofolic acid is shown to be a major form of folic acid in rat liver, along with N5-methyltetrahydrofolic acid. A system of differential microbiological assays that will identify the folates occurring naturally in tissues is presented. It involves rapid heat inactivation of natural enzymes, cleavage of folate polyglutamates with conjugase, and assay with three microorganisms under standardized conditions that allow identification and quantitation of both free and conjugated forms of four folate derivatives. Evidence is presented that the procedures outlined produce a minimum of change in the naturally occurring folate derivatives.