The Mycobacterium tuberculosis (MTB) SigF alternate sigma factor has been shown to have significant homology to the Bacillus subtilis (BSU) stress-response sigma factor, SigB, as well as to the BSU developmental sigma factor, SigF. In this study we report that like both the BSU sigB and sigF genes, MTB sigF is preceded by an open reading frame (usfX) encoding a protein with significant homology to the previously described BSU anti-sigma factors, RsbW and SpollAB. Sequence analysis suggests that the usfX and sigF genes appear to be cotranscribed and translationally coupled. A second open reading frame called usfY precedes usfX, but has no significant homologues and may not be contranscribed with the usfX and sigF. The sigF gene has been overexpressed in Escherichia coli, purified, and used to raise polyclonal antibodies. Immunoblotting demonstrates that MTB SigF is antigenically closer to BSU SigB than to BSU SigF. Fusion of the MTB sigF gene to the MTB hsp60 promoter has demonstrated that inappropriate overexpression of sigF is lethal for the slow-grower Mycobacterium bovis bacille Calmette-Guérin (BCG), but not for the rapid-grower Mycobacterium smegmatis which lacks a sigF homologue. Hence, sigF, encoding an MTB stress response, stationary phase transcription factor, is preceded by an antisigma factor homologue and is incompatible with growth when constitutively overexpressed in BCG.