Most of the conventional wax D preparations from human strain Mycobacterium tuberculosis induced adjuvant arthritis (AA) and typical delayed skin reaction to purified protein derivative (PPD) in the rat. However, the tuberculin-sensitizing ability of these waxes D as judged by skin testing with PPD was variable from preparation to preparation. “Pure” wax D containing trace amounts of tuberculin-sensitizing materials has recently been extracted from a human strain M. tuberculosis H37Ra, cultivated on Sauton medium for 4 weeks. The water-soluble portion (WSP) subfractions designated as F-IA and F-IB were obtained from this pure wax D. These subfractions, which are homogeneous as revealed by Ouchterlony tests and immunoelectrophoresis, were capable of inducing AA and a delayed hypersensitivity elicited by themselves, but not by PPD. PPD and π, which were less immunogenic than F-IA and F-IB, failed to produce AA. Arthritogenicity of the parent wax D correlated more closely with the degree of skin reaction to the WSP subfractions than with the PPD skin reaction. However, after development of AA the skin reaction to F-IB decreased significantly, especially in severely diseased rats, whereas PPD positivity remained constant and strong. Although Ouchterlony double diffusion tests with F-IA, F-IB, PPD and π revealed that an identical or partially identical component is present in all, F-IA and F-IB are peptidopolysaccharides and differ in their immunologic behavior from the tuberculoproteins, PPD and π. Evidence of two different delayed-type antigens in conventional wax D as opposed to one in pure wax D was revealed in Ouchterlony tests.