The genus Rhodococcus exhibits a broad range of catalytic activity and is tolerant to various kinds of organic solvents. This property makes rhodococci suitable for use as a whole-cell catalyst. Various tools for genetic engineering have been developed to use Rhodococcus erythropolis as a host for bioconversion. In this study, we investigated the protein expression responses of R. erythropolis strains and found that isocitrate lyase production in R. erythropolis PR4 (ICL Re ) was induced by methanol. By analyzing the regulation mechanisms of icl Re expression, the ~200-bp upstream region from the first nucleotide of the translation initiation codon of icl Re was shown to be sufficient for the methanol-inducible expression. Also, the ~100-bp upstream region exhibited strong constitutive promoter activity by an unknown mechanism(s). By investigating proteins that bound to the upstream region of icl Re in vitro, a RamB homologue of R. erythropolis PR4 (RamB Re ) was identified. Moreover, 2 putative RamB Re binding sites were identified in the upstream region of icl Re through pull-down assays. A ramB Re knockout experiment suggested that RamB Re negatively controlled the expression of icl Re and that RamB Re regulation was dependent on the availability of a carbon source. On the basis of these findings, we were able to create novel methanol-inducible and strong constitutive expression vectors.