When isolated rat hearts were perfused with medium containing 3-O-methyl-D-glucose (3MG) a compound with the chromatographic behavior expected for 3MG-6-phosphate was formed. This assignment is supported by the action of alkaline phosphatase and by production of an identical peak on incubation of 3MG with yeast hexokinase in a buffer containing adenosine triphosphate and Mg++ ions. The clearance of labeled 3MG from isolated perfused rat hearts was biphasic and eventually total. Since sugar phosphates do not cross cellular membranes, this behavior is consistent with both formation and dephosphorylation of 3MG-6-phosphate, with dephosphorylation rate-limiting the slow phase of the clearance. Phosphatase activity was also indicated by a decrease in 3MG-6-phosphate when hearts were not chilled and homogenized immediately after perfusion. The common assumption that 3MG is not metabolized after transport into cells is shown by these experiments to be erroneous.