The production of mannosidase activity by all currently recognized species of human viridans group streptococci was determined using an assay in which bacterial growth was dependent on the degradation of the high-mannose-type glycans of RNase B and subsequent utilization of released mannose. RNase B is an excellent substrate for the demonstration of mannosidase activity since it is a glycoprotein with a single glycosylation site which is occupied by high-mannose-type glycoforms containing five to nine mannose residues. Mannosidase activity was produced only by some members of the mitis group (Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii, Streptococcus cristatus, Streptococcus infantis, Streptococcus parasanguinis, and Streptococcus pneumoniae) and Streptococcus intermedius of the anginosus group. None of the other species within the salivarius and mutans groups or Streptococcus peroris and Streptococcus sanguinis produced mannosidase activity. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, it was demonstrated that the Man(5) glycan alone was degraded while Man(6) to Man(9), which contain terminal alpha(1-->2) mannose residues in addition to the alpha(1-->3), alpha(1-->6), and beta(1-->4) residues present in Man(5), remained intact. Investigations on mannosidase production using synthetic (4-methylumbelliferone- or p-nitrophenol-linked) alpha- or beta-mannosides as substrates indicated that there was no correlation between degradation of these substrates and degradation of the Man(5) glycan of RNase B. No species degraded these alpha-linked mannosides, while degradation of the beta-linked synthetic substrates was restricted to strains within the Streptococcus anginosus, S. gordonii, and S. intermedius species. The data generated using a native glycoprotein as the substrate demonstrate that mannosidase production within the viridans group streptococci is more widely distributed than had previously been considered.