Background Adenosine deaminase (ADA) catalyzes hydrolytic and irreversible deamination of deoxyadenosine into deoxyinosine and of adenosine into inosine, and is related to lymphocytic proliferation and differentiation. The measurement of ADA activity in body fluids is a useful tool in the evaluation of mycobacterial infections. Elevated ADA activity has been found in pleural effusions of patients with pleural tuberculosis relative to those from patients with nontuberculous pleural diseases, and is mainly associated with cellular host factors such as monocyte-macrophages or lymphocytes. In contrast, there is little information about ADA activity measurement in mycobacteria culture supernatants. Methods We evaluated ADA activity as described by Giusti in the culture supernatants of eight Mycobacterium tuberculosis isolates. Results Mycobacteria culture supernatants did not display any ADA activity. Conclusions This result supports the notion that Mycobacterium tuberculosis is not the source of ADA activity. However, increased ADA activity in biological fluids from tuberculosis patients might be due to the interaction of the mycobacterium with host factors.