The enzyme peptidyl-tRNA hydrolase (Pth, EC 220.127.116.11) is essential for the viability of bacteria. The ORF Rv1014c of Mycobacterium tuberculosis H37Rv, designated as the mtpth gene, was cloned and over-expressed and the product was purified. Generation of polyclonal antibodies against the purified recombinant protein, termed MtPth, facilitated detection of endogenously expressed MtPth in M. tuberculosis H37Rv cell lysate. MtPth could release diacetyl-[3H]-lysine from diacetyl-[3H]-lysyl-tRNALys with Michaelis-Menten kinetic parameters of Km=0.7±0.2 μM and kcat=1.22±0.2 s-1. Transformation of a pTrc99c/mtpth vector allowed the growth of E. coli thermosensitive Pth(ts) mutant strain AA7852 at the non-permissive temperature of 42°C, demonstrating the in vivo activity of MtPth. In addition, at 39°C, over-expression of MtPth in AA7852 cells allowed the cells to remain viable in the presence of up to 200 μg/ml erythromycin. A 3D fold based on NMR and a structural model based on the E. coli Pth crystal structure were generated for MtPth. The essential nature of conserved active-site residues N12, H22 and D95 of MtPth for catalysis was demonstrated by mutagenesis and complementation in E. coli mutant strain AA7852. Thermal and urea/guanidinium chloride (GdmCl)-induced unfolding curves for MtPth indicate a simple two-state unfolding process without any intermediates.