Tuberculosis remains a major health problem throughout the world and the failure of the existing bacille Calmette-Guérin (BCG) vaccine in recent trials has prompted a search for potential replacements. Recent advances in molecular and cell biology have cast doubts on the ability of genetic analysis alone to predict polygenic human diseases and other complex phenotypes and have therefore redirected our attention to proteome studies to complement information obtained from DNA sequencing initiatives. Novel acidic (pH 2.3-5) and basic (pH 6-11) IPG gel gradients were employed in conjunction with commercially available pH 4-7 gradients to significantly increase (fourfold) the number of protein spots previously resolved on two-dimensional (2-D) gels of Mycobacterium species. A total of 772 and 638 protein spots were observed for M. bovis BCG and M. tuberculosis H37Rv, respectively, the latter corresponding to only the pH regions 4-7 and 6-11. Of interest was the bimodal distribution observed for proteins separated from M. bovis BCG across both M(r) and pH ranges. Some differences in protein expression were observed between these two organisms, contrary to what may have been expected considering the high degree of conservation in gene order and sequence similarity between homologous genes. Further work will be directed towards a more detailed analysis of these differences, so as to allow more accurate diagnosis between vaccination and active tuberculosis. The latter is of major importance to epidemiological studies and for patient management.