The 40 kDa antigen of Mycobacterium tuberculosis is the first antigen reported to be present in the pathogenic M. tuberculosis , but not in the vaccine strain Mycobacterium bovis BCG. It is a functional L -alanine dehydrogenase (EC 220.127.116.11) and hence one of the few antigens possessing an enzymic activity. This makes the 40 kDa antigen attractive for potential diagnostic and therapeutic interventions. Recently, we developed a strategy to purify quantities of the recombinant protein in active form, and here we describe the biochemical properties of this enzyme. In the oxidative-deamination reaction, the enzyme showed K m values of 13.8 mM and 0.31 mM for L -alanine and NAD + , respectively, in a random-ordered mechanism. K m, app values in the reductive-amination reaction are 35.4 mM, 1.45 mM and 98.2 μ M for ammonium, pyruvate and NADH, respectively. The enzyme is highly specific for all of its substrates in both directions. The pH profile indicates that oxidative deamination virtually may not occur at physiological pH. Hence L -alanine most likely is the product of the reaction catalysed in vivo . The enzyme is heat-stable, losing practically no activity at 60 °C for several hours.