Chemical ionization mass spectra of several 16-membered basic macrolide antibiotics containing an amino sugar are described. Depending on the reagent gas used (methane, isobutane or ammonia), a common pattern is exhibited by the chemical ionization mass spectra of all the compounds. That is, they show the protonated molecular ion as the base peak, which clearly indicates the molecular weight. Fragmentation involving carbon-carbon bond fission rarely occurs. As the major fragmentation pathway, the glycosidic bond is cleaved at both sides of the glycosidic oxygen atom to produce two aglycone-ions and three sugar-derived ions. These are shown to be useful in the structural characterization of new macrolide antibiotics.