Methionine aminopeptidase (MetAP) carries out an essential posttranslational modification of nascent proteins by removing the N-terminal methionine and is a potential target for discovering antibacterial agents. To characterize and compare the two MetAPs in Mycobacterium tuberculosis, Rv0734 and Rv2861c genes encoding MetAPs from genome of Mycobacterium tuberculosis H37Rv were cloned and expressed in Escherichia coli. Comparative analysis showed that the two recombinant Mycobacterium tuberculosis MetAPs (MtMetAPs, with 6His-tag being cleaved) have different activities in their substrate specificity, divalent ion requirement, and temperature optima. The temperature for MtMetAP1a and MtMetAP1c were 55 and 37 degrees C, respectively, and MtMetAP1a was found to have good temperature stability. The activities of MtMetAPs were increased by Co2+ ions, but were strongly inhibited by Cu2+, Fe2+, and Ni2+. In addition, the MtMetAP1a and MtMetAP1c activities were stimulated by Mg2+ and Zn2+, respectively. Transcriptional comparative analysis of these two genes revealed that, in both H37Ra and H37Rv, there was approximately a 2-fold decrease of Rv0734 transcripts in 60-day-old stationary phase culture comparing to that in 14-day-old log phase bacilli. On the other hand, the transcription level of Rv2861c in tested mycobacteria in log phase culture was nearly 1.5 times lower than that in stationary phase culture. The result suggests that the two MtMetMAPs may perform important function in different growth phases of Mycobacterium tuberculosis.