The maltose regulon of Escherichia coli comprises several operons that are under common regulatory control of the MalT activator protein. Five mal genes, organized in two divergent operons, code for a binding-protein-dependent transport system specific for maltose and maltodextrins. MalK, one of the subunits of this transport system, not only is essential for transport but also plays a role in regulation. Mutations abolishing MalK function not only result in inability to transport maltose but also cause constitutive expression of the maltose regulon. For this constitutivity to be exerted, the function of an additional gene product, MalI, is necessary. Using the constitutive expression of a malK-lacZ fusion as a signal, we cloned the malI gene, expressed it in minicells, and determined its DNA sequence. The sequence predicted a protein of 34,729 molecular weight, in agreement with the apparent molecular weight of the protein (35,000) when expressed in minicells and analyzed by polyacrylamide gel electrophoresis and autoradiography. MalI exhibited high homology to the repressor proteins GalR, CytR, and LacI. When the amino acid sequences were appropriately aligned, MalI showed 28% identity to GalR, 21% to CytR, and 24% to LacI. Including conservative amino acid exchanges, these numbers increased to 69, 56, and 58%, respectively. The regions of high homology were clustered in particular at the N-terminal portion of the protein that includes the helix-turn-helix motif thought to be involved in DNA binding. The protein contained a short stretch of 30 amino acids that was surprisingly homologous to a sequence in MalT. The amino-terminal half of the protein exhibited significant homology with MalK. The transcriptional start of malI was determined by reverse transcriptase and by S1 nuclease mapping. We found a possible binding site for cyclic AMP receptor protein in the promoter region of malI as well as two perfect direct repeats of 14 base pairs with twofold symmetry indicating their possible role as operator sites. Upstream to malI we observed a divergent open reading frame that extended to the end of the sequenced DNA.