Genes acrAB encode a multidrug efflux pump in Escherichia coli. We have previously reported that transcription of acrAB is increased under general stress conditions (i.e. 4% ethanol, 0.5 M NaCl, and the stationary phase in Luria-Bertani medium). In this study, lacZ transcriptional fusions and an in vitro gel mobility shift assay have been utilized to study the mechanisms governing the regulation of acrAB. We found that a closely linked gene, acrR, encoded a repressor of acrAB. Nevertheless, the general stress conditions increased transcription of acrAB in the absence of functional AcrR, and such conditions surprisingly increased the transcription of acrR even more strongly than that of acrAB. These results suggest that the general-stress-induced transcription of acrAB is primarily mediated by global regulatory pathway(s), and that one major role of AcrR is to function as a specific secondary modulator to fine tune the level of acrAB transcription and to prevent the unwanted overexpression of acrAB. To our knowledge, this represents a novel mechanism of regulating gene expression in E. coli. Evidence also suggests that the up-regulation of acrAB expression under general stress conditions is not likely to be mediated by the known global regulators, such as MarA or SoxS, although elevated levels of these proteins were shown to increase the transcription of acrAB.