Because many membrane-associated proteins represent potential drug targets, diagnostic probes, and components of vaccines, we have chosen to study the membrane proteins of Mycobacterium tuberculosis H37Rv. To remove cytosolic proteins and facilitate access to the integral membrane proteins, membrane fractions of M. tuberculosis H37Rv were intensely washed with 5 M urea and high pH carbonate solution. One-dimensional SDS-PAGE, followed by enzymatic hydrolysis and nanoLC electrospray ionization MS/MS, proved to be the most efficient way to identify the proteins contained within the membrane fraction. Here we report 349 protein identifications in total, validated by at least two tryptic peptide matches and MOWSE scores greater than 75. Of those 349 proteins, 100 are integral membrane proteins with at least one predicted transmembrane alpha helix (excluding the possible signal sequence). 84 M. tuberculosis H37Rv proteins, including 42 integral membrane proteins, are described for the first time.