The LLC-PK1 cell line has been well characterized concerning its proximal tubule-like Na+-dependent active sugar transporter in the apical membrane. In this study, we investigated the uptake of the glucose analogue, 2-deoxy-D-glucose (2DOG), a paradigm substrate for the facilitated diffusion, Na+-independent sugar transporter in the renal basolateral membrane. The uptake of 0.1 mM 2-[14C]DOG by confluent LLC-PK1 cell sheets at 25 degrees C is linear at least to 10 min, at which time greater than 90% of intracellular radioactivity is 2DOG phosphate. The uptake of this analogue by LLC-PK1 cells is Na+ independent, and the transporter appears to be localized to the basolateral cell membrane. Phlorizin is a much less effective inhibitor than its aglycon, phloretin. Cytochalasin B is also an effective inhibitor, but it causes morphological changes in the cells at concentrations required to inhibit transport. Specificity studies indicate that this transport system requires a hexose with a free hydroxyl at C-1, and that the hydroxyls at C-3 and C-4 be preferably in the equatorial position. Glucose starvation causes an increased rate of 2DOG uptake. Subconfluent (cycling) cultures of LLC-PK1 cells have a threefold greater rate of 2DOG uptake than that seen in confluent (noncycling) LLC-PK1 cells.