The synthesis of 5,11-methenyltetrahydrohomofolate was accomplished by treatment of tetrahydrohomofolate (H4homofolate) with triethyl orthoformate in glacial acetic acid. This compound is a homofolate analogue of 5,10-methenyltetrahydrofolate which serves as one precursor to the 10-formyl one-carbon donor for the first transformylation in de novo purine biosynthesis, namely, the conversion of glycinamide ribonucleotide (GAR) to N-formylglycinamide ribonucleotide (FGAR), catalyzed by the enzyme glycinamide ribonucleotide transformylase (EC 2.1.2.2). The analogue proved to retard the rate of formation of formylglycinamide ribonucleotide apparently by inhibiting the rate of synthesis of 10-formyltetrahydrofolate, the actual cofactor for the transformylase enzyme, from 5,10-methenyltetrahydrofolate. Its inhibition of the enzyme, 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), was competitive against (+)-L-5,10-methenyltetrahydrofolate, with a Ki = 41 micro M. This derivative of homofolate may be responsible for its inhibition of purine biosynthesis in Sarcoma 180 cells.