Glucose transport was studied in marine mussels of the genus Mytilus. Initial observations, with intact animals and isolated gills, indicated that net uptake of glucose occurred in mussels by a carrier-mediated, Na+-sensitive process. Subsequent studies included use of brush-border membrane vesicles (BBMV) in order to characterize this transport in greater detail. The highest activity of Na+-dependent glucose transport was found in the brush-border membrane fractions used in this study, while basal-lateral membrane fractions contained the highest specific binding of ouabain. Glucose uptake into BBMV showed specificity for Na+, and concentrative glucose transport was observed in the presence of an inwardly directed Na+ gradient. There was a single saturable pathway for glucose uptake, with an apparent Kt of 3 microM in BBMV and 9 microM in intact gills. The kinetics of Na+ activation of glucose uptake were sigmoidal, with apparent Hill coefficients of 1.5 in BBMV and 1.2 in isolated gills, indicating that more than one Na+ may be involved in the transport of each glucose. Harmaline inhibited glucose transport in mussel BBMV with a Ki of 44 microM. The uptake of glucose was electrogenic and stimulated by an inside-negative membrane potential. The substrate specificity in intact gills and BBMV resembled that of Na+-glucose cotransporters in other systems; D-glucose and alpha-methyl glucopyranoside were the most effective inhibitors of Na+-glucose transport, D-galactose was intermediate in its inhibition, and there was little or no effect of L-glucose, D-fructose, 2-deoxy-glucose, or 3-O-methyl glucose. Phlorizin was an effective inhibitor of Na+-glucose uptake, with an apparent Ki of 154 nM in BBMV and 21 nM in intact gills. While the qualitative characteristics of glucose transport in the mussel gill were similar to those in other epithelia, the quantitative characteristics of this process reflect adaptation to the seawater environment of this animal.