The Mycobacterium tuberculosis prrA-prrB (Rv0903c-Rv0902c) two-component regulatory system is expressed during intracellular growth in human macrophages and is required for early intracellular multiplication in murine macrophages, suggesting its importance in establishing infection. To better understand the function of the prrA-prrB two-component system, we defined the transcriptional characteristics of the prrA and prrB genes during exponential and stationary growth and upon exposure to different environmental stresses and attempted to generate a prrA-prrB deletion mutant. The prrA and prrB genes constitute an operon and are cotranscribed during logarithmic growth, with transcriptional levels decreasing in stationary phase and during hypoxia. Despite the transcriptional differences, PrrA protein levels remained relatively stable throughout growth and in hypoxia. Under conditions of nitrogen limitation, prrAB transcription was induced, while acidic pH stress and carbon starvation did not significantly alter transcript levels. Deletion of the prrAB operon on the chromosome of M. tuberculosis H37Rv occurred only in the presence of an episomal copy of the prrAB genes, indicating that this two-component system is essential for viability. Characterization of the prrAB locus in M. tuberculosis Mt21D3, a previously described prrA transposon mutant, revealed that this strain is not a true prrA knockout mutant. Rather, Tn5367 transposon insertion into the prrA promoter only decreased prrA and prrB transcription and PrrA levels in Mt21D3 compared to those in the parental Mt103 clinical strain. These data provide the first report describing the essentiality of the M. tuberculosis prrAB two-component system and reveal insights into its potential role in mycobacterial growth and metabolism.