The acetate replacing activity of DL-α-lipoic acid used for the growth of Streptococcus faecalis R was markedly affected by the composition of the medium. When a semisynthetic basal medium supplemented with casamino acid (Difco) as an amino acid source was used, the ability of DL-α-lipoic acid to replace acetate was greatly enhanced in the presence of sodium citrate or pyruvate. In the absence of the substances, however, DL-α-lipoic acid has no activity to support the growth of St. faecalis R. Such activity enhancing effect of sodium citrate and pyruvate was not observed when a synthetic medium was used. In the synthetic medium, DL-α-lipoic acid acted for acetate without help of other substances. From the results mentioned above, two assay methods of DL-α-lipoic acid using St. faecalis R were proposed. The first is to calculate from the acetate replacing activity in the semisynthetic medium supplemented with sodium citrate. The second consists of utilizing the antagonistic activity of DL-α-lipoic acid to the growth inhibiting activity of propionic acid. In the latter, the assay medium was that described by Stokstad but modified a little. In this simplified medium, the irreguler response of St. faecalis R which was observed occasionally was fairly diminished.