A novel immunogenic antigen, CFP 6 was purified from culture filtrate of M. tuberculosis by a preparatory 2-D electrophoresis method. The protein focused at pI of 4.0 during isoelectric focusing. Molecular weight of the purified protein by ES MS was found to be 11.61 kD. N-terminal amino acid sequence of CFP 6 could be aligned to the deduced amino acid sequence from ORF Rv3004 and was found to be a novel protein with 112 aa residues. Single N-terminal sequence showed that the purified protein was essentially free from contaminants and the amino acid analysis of the antigen was in good agreement with the DNA sequence deduced amino acid composition. Purified CFP 6 was studied for its ability to induce proliferative responses of peripheral blood lymphocytes from five categories of human subjects. These were: untreated, active pulmonary tuberculosis patients; patients after 2-3 months of chemotherapy; vaccinated professional contacts; vaccinated/nonvaccinated healthy controls. CFP 6 elicited high proliferative responses in healthy contacts and patients recovering from the disease. This protein also induced the release of a significantly high amount of IFN-gamma in cell culture supernatant of healthy contacts as compared to other categories of subjects. This protein was further evaluated and compared with PPD and total CS for its DTH inducing ability in guinea pigs immunised with BCG or M. tuberculosis H(37)Rv. CFP 6 elicited a powerful immune response in vitro and in vivo animal model, hence seems to be an immunologically important protein.