Abstract: A new test substance for use in skin testing for tuberculosis, which substance is produced from Mycobacterium tuberculosis (M.tb.) culture filtrate or bacillary extract. The culture filtrate or extract is first fractionated by means of chromatography. Resultant fractions are then further fractionated so as to produce subfractions which contain at least one M.tb. specific antigen and are substantially free of antigens crossreactive with atypical mycobacteria. At least two of these subfractions may then be combined to produce a mix or new test substance, containing a plurality of M.tb. specific antigens only, capable of stimulating lymphocytes in individuals previously sensitized to M. tuberculosis antigens.