The gabCTDP gene cluster, which specifies and regulates synthesis of the gamma-aminobutyrate (GABA) transport carrier, of glutamate-succinic semialdehyde transaminase, and of succinic semialdehyde dehydrogenase, responsible for the uptake and metabolism of gamma-aminobutyric acid in Escherichia coli K-12, was cloned in vivo, using the mini-Mu replicon bacteriophage Mu dI5086 as the vector. A subclone containing a 7.8-kilobase (kb) EcoRI-HindIII fragment complemented all of our Gab- mutants. By restriction mapping, this DNA fragment was located at kb 2800.5 to 2808.5 on the physical map of the E. coli K-12 chromosome. A subclone containing a 1.8-kb EcoRI-SalI fragment complemented the gab-repressed strain CS101A (wild-type gabC) but did not complement any gab structural gene mutants. The gab genes are divergently transcribed from promoters located in the vicinity of the unique BamHI site. Transcription in both directions is under dual control of catabolite repression and nitrogen regulation. Using a procaryotic DNA-directed translation system, we observed three insert-coded polypeptide bands of 53 to 55, 45 to 48, and 40 to 43 kilodaltons (kDa). In vivo studies with subcloned fragments of the gab DNA identified the 53- to 55- and 45- to 48-kDa bands as products of the BamHI-SalI fragment and the 40- to 43-kDa band as the product of the EcoRI-SalI fragment. An additional 26- to 28-kDa band was identified as the product of the BamHI-HindIII fragment. Furthermore, the BamHI-SalI fragment was shown to specify synthesis of the two GABA enzymes, whereas synthesis of the GABA carrier was specified by the BamHI-HindIII fragment. No catalytic function in addition to its regulatory role could be attributed to the EcoRI-SalI gene product.