In recent years, tuberculosis has risen in incidence to become the single most deadly infectious disease, killing approximately 3 million people globally each year. This is partly due to the almost extraordinary ability of the causative agent, Mycobacterium tuberculosis, to overcome antibiotics by developing resistance at the genetic level. This leaves the worldwide biochemical community scrambling to engineer new antibiotics with novel targets, rendering them capable of thwarting bacilli immune to the drugs currently in use. In an effort to contribute to this international quest, here we have focused on one biochemical pathway within M. tuberculosis believed to have potential as an antibiotic target: the de novo lipoic acid biosynthesis pathway. For each protein involved in this pathway - acyl-carrier protein (ACP), octanoyltransferase (LipB), and lipoyl synthase (LipA) - the corresponding gene was cloned. LipA was chosen for further analysis, and was subsequently expressed, purified, and characterized biochemically. Herein we have provided evidence to suggest that MtLipA is analogous to EcLipA with regard to its 2 [4Fe-4S]2+ cluster catalytic center and its basic activity in the presence of substrate.