The effects of the hallucinogen harmaline on D-glucose, L-alanine, and Na+ transport were studied in microvillus membrane vesicles isolated from the rabbit renal cortex. Harmaline had no effect on glucose transport in the absence of Na+, but reversibly inhibited sugar flux in the presence of NaCl. Inhibition of Na+-dependent glucose transport was inversely related to the Na+ concentrations. The hallucinogen competitively inhibited the Na+ activation of phlorizin binding to the membranes but did not inhibit phlorizin binding in the absence of Na+. Harmaline inhibited Na+-dependent alanine transport and, at higher drug concentrations, the amino acid flux in the absence of NaCl. Harmaline competitively inhibited the rate of Na+ uptake which, in the absence of glucose and alanine, is known to occur via Na+-H+ exchange. The hallucinogen trans-inhibited the efflux of glucoe and Na+ from membrane vesicles preloaded with the solutes. These findings suggest that harmaline is a direct inhibitor of microvillus membrane transport processes and acts as a competitive inhibitor of Na+ transport sites. Harmaline may therefore be a useful investigative tool for studying mechanisms of Na+-coupled transport in the luminal membrane of the proximal tubular cell.