An N-acetyl-β-d-glucosaminidase (GcnA) from Streptococcus gordonii FSS2 was cloned and sequenced. GcnA had a deduced molecular mass of 72,120 Da. The molecular weight after gel-filtration chromatography was 140,000 Da and by SDS–PAGE was 70,000 Da, indicating that the native protein was a homodimer. The deduced amino acid sequence had significant homology to a glycosyl hydrolase from Streptococcus pneumoniae and the conserved catalytic domain of the Family 20 glycosyl hydrolases. GcnA catalysed the hydrolysis of the synthetic substrates, 4-methylumbelliferyl (4MU)-N-acetyl-β-d-glucosaminide, 4MU-N-acetyl-β-d-galactosaminide, 4-MU-β-d-N,N′-diacetylchitobioside, and 4-MU-β-d-N,N′,N″-chitotrioside as well as the respective chito-oligosaccharides. GcnA was optimally active at pH 6.6 and 42 °C. The Km for 4-MU-β-d-N,N′,N″-chitotrioside, 45 μM, was the lowest for all the substrates tested. Hg2+, Cu2+, Fe2+, and Zn2+ completely inhibited while Co2+, Mn2+, and Ni2+ partially inhibited activity. S. gordonii FSS2 and a GcnA negative mutant grew equally well on chito-oligosaccharides as substrates. The S. gordonii sequencing projects indicate two further N-acetyl-β-d-glucosaminidase activities. [Copyright &y& Elsevier]