A highly purified tuberculin peptide, named TPH71U, was isolated from the heated culture filtrate of Mycobacterium tuberculosis strain H37Rv. This peptide was prepared by precipitation with ammonium sulfate and by treatment with diethylaminoethyl-Sephadex, Sephadex G-75, and preparative gel electrophoresis in the presence of a high concentration of urea. The presence of urea remarkably increased the sensitivity of the gel analysis and the efficiency of the preparation procedure. The isolated peptide, TPH71U, was homogeneous in the gel electrophoretic analysis, and the molecular weight was estimated to be 5,800 daltons. The amino acid composition was analyzed. The peptide elicited strong tuberculin cantaneous reactions in guinea pigs sensitized with Mycobacterium tuberculosis strains H37Rv and Aoyama B, and Bacille Calmette Guèrin, and weak reactions in those sensitized with Mycobacterium intracellulare, Mycobacterium kansasii, and Mycobacterium phlei. Based on the cutaneous reactions to this peptide, its potency was estimated to be approximately one twentieth of that of the standard tuberculin purified protein derivative.