The active transport of methyl beta-D-galactoside and some other analogs of D-glucose and D-galactose was studied in slices of rabbit and renal cortex. 1. The non-metabolizable methyl beta-D-galactoside accumulates in renal cortical cells against its concentration gradient. At 1 mM substrate concentration (O2, 35 degrees C, 60 min incubation) the gradient was 2.36 +/- 0.11 S.E. (n = 33). The Kt was 1.50 +/- 0.02 mM. The active transport of the substrate was inhibited by dinitrophenol, phlorizin, absence of Na+ and by ouabain. This inhibition was incomplete, suggesting that the sugar may enter the cells by two separate pathways, only one of which was coupled to the down-hill electrochemical Na gradient. 2. The structural requirements for the interaction between substrate and the carrier were defined: (a) by testing the transport behavior of some analogs (1,5-anhydro-D-glucitol; methyl beta-thio-D-galadtoside; 3-deoxy-D-glucose; 4-deoxy-D-glucose; 5-thio-D-glucose; 6-deoxy-D-glucose and methyl-alpha-6-deoxy-D-glucoside); and (b) by inhibition analysis of methyl beta-D-galactoside transport. The role of each hydroxyl of the sugar molecule was tested by using a total of 41 analogs modified on each C by replacing -OH by -H, -O-CH3, -F and in some instances also by -SH. 3. The carrier is shared by D-glucose, D-galactose and their methyl glycosides. A pyranose ring is mandatory. The D-glucoconfiguration is preferred for the interaction with the carrier. 4. Replacement of -OH by -H or -S practically abolished (on C1, C2, C3) or greatly reduced (on C4) the affinity of the analog for the carrier. This was also confirmed by demonstrating that 1-deoxy-, and 3-deoxy-glucose and the thio-galactoside were not actively transported and their entry into the cells was not markedly affected by phlorizin, dinitrophenol, ouabain or absence of Na+. 4-Deoxy-D-glucose was taken up and its transport was inhibited by agents affecting the transport of methyl beta-D-galactoside. 5. Replacement of -OH by -F did not abolish the affinity of the analogs for the carrier, indicating hydrogen bonding between the carrier and the oxygens at C1, C2, C3, and C4. 6. 5-Thio-D-glucose was not transported against its concentration gradient and also poorly interacted with the carrier as shown by inhibition analysis. Hydrogen bonding between the carrier and the pyranose ring oxygen is suggested. 7. 6-Deoxyglucose is a potent inhibitor of methyl beta-D-galactoside transport although it is not actively taken up by the tissue. It is concluded that a hydroxyl at C6 is required for transport, but is not mandatory for an interaction with the carrier. However, 6-deoxy-D-galactose was ineffective as inhibitor. 8. The specificity of the carrier involved in the renal active transport of D-glucose, D-galactose and their methyl glycosides resembles qualitatively, and mostly also quantitatively that described for intestinal transport of these sugars.