The 19-kDa glycolipoprotein of Mycobacterium tuberculosis (PT19) is a prominent antigen recognized by both T cells and antibodies from tuberculosis patients. We report here that two strains, I2646 and S1, when grown either in bacteriological culture or during infection of mice, do not produce this constituent, as judged by ELISA and Western blot assays. Southern blot analysis of the chromosomal DNA showed that both strains displayed the restriction fragment as in H37Rv DNA, suggesting the lack of gross gene alterations. Sequence analysis revealed multiple microlesions including small deletions, point mutations and nucleotide insertions, leading to either premature termination or alteration of open reading frame in both strains. Transformation of both mutant strains with the wild-type gene on a multicopy plasmid resulted in overproduction of native PT19. Infection of mice suggested that the I2646 is of low virulence and that the transformant-producing native PT19 exhibited higher virulence, as assessed by viable counts and gross lesions in the infected organs. The mechanisms and significance of the lack of PT19 production in certain M. tuberculosis strains is discussed.