Tumors present a high glycolytic metabolism consuming more glucose than normal cells. Considering this property, some drugs have been tested to repress glycolytic flux and cellular proliferation. Recently, it was demonstrated that the alkylanting agent 3-Bromopyruvate (3BrPA) had a rapid and selective action in the death tumor process. Its targets of action have been related to the primary inhibition of glycolysis and ATP synthesis by mitochondria, acting directly on the hexokinase type II. This enzyme is the major isoform found in tumors and it is associated with mitochondria catalyzing the following reaction:  Glucose + ATP → Glucose-6-phosphate + ADP. However, the effects of 3BrPA on other types of hexokinases and on mitochondria are poorly known. The aim of this work was to investigate the effects of 3BrPA on the activities of hexokinase type I from mice brain (HK I), type II from human hepatoma cells (HepG2) (HK II) and glucokinase (GK) from mice liver. Different IC₅₀ values for 3BrPA inhibition were found under our assay conditions: 100 - 200mM for GK; > 1 mM for HK I; and a significant inhibition was observed for the HK II for HepG2 at 3BrPA concentration as higher as 5 mM at a reaction time of one hour. In addition, our results showed a 50 % reduction on viability and proliferation of HepG2 incubated for one hour with 0.1 to 1 mM of 3BrPA. Surprisingly, a dose as low as 50 mM of 3BrPA was sufficient to inhibit almost completely oxygen consumption by these cells. These results suggest that 3BrPA acts in different steps of glycolysis or, more importantly, the mitochondria respiration is the primary site of action of this drug. More studies are necessary to evaluate the effects of 3BrPA on different hexokinases types and its action on mitochondria.