The 5-kDa protein PorA of the Gram-positive bacterium Corynebacterium glutamicum is the subunit of the cell wall channel. Antibodies raised against PorA specifically detected the protein on the cell surface. PorA was sequenced using Edman degradation and a gas phase sequencer. The primary sequence was used to create degenerate oligonucleotide primers. The gene of the channel-forming protein and its flanking regions were obtained by PCR followed by inverse PCR. The gene porA comprises 138 bp and encodes a 45amino-acid-long acidic polypeptide with an excess of four negatively charged amino acids in agreement with the high cation selectivity of the PorA cell wall channel. PorA does not contain an N-terminal extension. A ribosomal-binding site was recognized 6 bp before the start codon ATG of porA. It codes for the smallest subunit of a membrane channel known so far and for the first cell wall channel protein of a corynebacterium. Southern blots demonstrated that only the chromosomes of corynebacteria contain homologous sequences to porA; no hybridization could be detected with DNA from other mycolata. q FEBS 2001 Cloning and sequencing of porA of C. glutamicum (Eur. J. Biochem. 268) 463 q FEBS 2001 Cloning and sequencing of porA of C. glutamicum (Eur. J. Biochem. 268) 467 q FEBS 2001 Cloning and sequencing of porA of C. glutamicum (Eur. J. Biochem. 268) 469