Salmonella typhimurium has the capacity to enter into and multiply within epithelial cells. During the entire intracellular stage, bacteria are enclosed within a vacuole. To characterize the micro–environment of the bacteria–containing vacuoles, we have used a new method to measure the expression levels of several S. typhimurium genes in intracellular bacteria within Madin–Darby canine kidney (MDCK) epithelial cells. Our study was based on the determination of ß–galactosidase activity derived from lacZ transcriptional fusions using the highly sensitive substrate fluorescein–di–ß–D–galactoside (FDG). Expression of the iroA and mgtB genes (induced by Fe2+ and Mg2+ limitation respectively), and cadA (induced by pH 6.0 in the presence of lysine, with enhanced expression under anaerobiosis) were characterized at different post–infection times. High intracellular expression levels were detected for the iroA and mgtB genes, suggesting that the concentrations of free Fe2+ and Mg2+ in the vacuole may be low. cadA actitvity was detected only at early post–infection times (4 h), suggesting that the vacuole may have a mild–acidic pH, and oxygen and lysine present at this time. Globally, the results reported indicate that the use of a highly sensitive ß–galactosidase substrate can provide information about the micro–enviroment within which an intracellular pathogen, such as S. typhimurium, resides.