Kinetic and optical studies were performed on the reductive half-reaction of soluble, quinoprotein glucose dehydrogenase (sGDH), i.e., on the conversion of sGDHox plus aldose sugar into sGDHred plus corresponding aldonolactone. It appears that the nature and stereochemical configuration of the substituents at certain positions in the aldose molecule determine the substrate specificity pattern: absolute specificity exists with respect to the C1-position (only sugars being oxidized which have the same configuration of the H/OH substituents at this site as the beta-anomer of glucose, not those with the opposite one) and with respect to the overall conformation of the sugar molecule (sugars with a 4C1 chair conformation are substrates, those with a 1C4 one are not); the nature and configuration of the substituents at the 3-position are hardly relevant for activity, and an equatorial pyranose group at the 4-position exhibits only aspecific hindering of the binding of the aldose moiety of a disaccharide. The pH optimum determined for glucose oxidation appeared to be 7.0, implying that reoxidation of sGDHred is rate-limiting with those electron acceptors displaying a different value under steady-state conditions. The kinetic mechanism of sGDH consists of (a) step(s) in which a fluorescing intermediate is formed, and a subsequent, irreversible step, determining the overall rate of the reductive half-reaction. The consequences of this for the likeliness of chemical mechanisms where glucose is oxidized by covalent catalysis in which a C5-adduct of glucose and PQQ are involved, or by hydride transfer from glucose to PQQ, followed by tautomerization of C5-reduced PQQ to PQQH2, are discussed. The negative cooperative behavior of sGDH seems to be due to substrate-occupation-dependent subunit interaction in the dimeric enzyme molecule, leading to a large increase of the turnover rate under saturating conditions.