Microorganisms have the capacity to sense their environment and to respond to it by alteration in gene expression and protein synthesis. Two-dimensional electrophoresis (2-DE) provides a powerful tool to examine the global response in bacterial protein synthesis upon exposure to different environmental signals. One of the most complex environments encountered by facultative intracellular pathogenic bacteria is the intracellular environment of the host cell. Numerous studies have documented that intracellular bacterial pathogens that replicate within phagosomes are simultaneously exposed to multiple signals and they respond to them by a global alteration in protein synthesis that involves elevated levels of several stress-induced proteins. This stress response is manifested regardless of the nature or the stage of maturation of the phagosome of different intracellular pathogens. In contrast, intracellular bacterial pathogens that replicate within the cytoplasm undergo phenotypic modulation in response to the cytoplasmic environment, but their responses do not include elevated levels of stress-induced proteins. This review describes the use of 2-DE to examine bacterial phenotypic modulation in response to the intracellular environment and contrasts this response between three intracellular pathogens; Legionella pneumophila, Salmonella typhimurium, and Listeria monocytogenes. The Legionella pneumophila phagosome is completely blocked from maturation through the endosomal lysosomal pathway but the S. typhimurium phagosome is a specialized compartment that has partial characteristics of an acidified late endosome, while L. monocytogenes rapidly escapes from an acidified phagosome into the cytoplasm.