The effect of dimethyl sulfoxide (DMSO) on the rate of glucose oxidation by cultured rat glomerular mesangial cells, human erythrocytes and peritoneal exudate cells was studied. Mesangial cells, erythrocytes and peritoneal exudate cells incubated with DMSO showed enhancement of 14CO2 production from D-[1-14C] glucose but not from D-[6-14C] glucose. The concentration of DMSO required to stimulate respiratory burst activity was lowest for erythrocytes and highest for peritoneal exudate cells. Studies utilizing tritiated deoxyglucose revealed that the increased glucose oxidation associated with DMSO exposure was not due to increased transmembrane glucose movement at low concentrations of DMSO, and only partially responsible at high concentrations of DMSO. This study documents the ability of DMSO to specifically enhance the activity of the hexose monophosphate shunt pathway in all cells studied. The precise mechanism whereby DMSO stimulates shunt activity remains unknown.