Glucose is transported across the cell membrane of Neurospora crassa by two physiologically and kinetically distinct transport systems. System II is repressed by growth of the cells in 0.1 m glucose. System I is synthesized constitutively. The apparent K(m) for glucose uptake by system I and system II are 25 and 0.04 mm, respectively. Both uptake systems are temperature dependent, and are inhibited by NaN(3) and 2,4-dinitrophenol. Glucose uptake by system II was not inhibited by fructose, galactose, or lactose. However, glucose was shown to be a noncompetitive inhibitor of fructose and galactose uptake. The transport rate of [(14)C]3-0-methyl-d-glucose (3-0-MG) was higher in cells preloaded with unlabeled 3-0-MG than in control cells. The rate of entry of labeled 3-0-MG was only slightly inhibited by the presence of NaN(3) in the medium. Further, NaN(3) caused a rapid efflux of accumulated [(14)C]3-0-MG. These data imply that the energetic step in the transport process prevents efflux.