1. Fluorescence imaging of antibodies was used to show that phorbol 12-myristate 13-acetate (PMA) induces a 4-fold increase in the amount of hexokinase relative to the control in the cortical shell of rat peritoneal macrophage cytosol adjacent to the plasma membrane, and a corresponding depletion in the amount of hexokinase in the central core of the cytosol. However, there was no significant PMA-dependent change in the distribution of glucose-6-phosphate dehydrogenase. 2. Cytochalasin D, an inhibitor of actin microfilament polymerization, prevented the PMA-induced hexokinase translocation and also reduced the PMA-dependent increases in 2-deoxy-D-glucose transport and glucose-dependent PMA-stimulated superoxide production. 3. PMA caused a contraction of the width of the cortical F-actin zone. Cytochalasin D caused some dispersal of F-actin within the cell, increasing the density of F-actin within the central cytosolic core and causing aggregation of the F-actin within the cortex. These data are consistent with the view that PMA induces attachment of hexokinase to microfilaments within the cortical zone adjacent to the cell membrane of macrophages, and cytochalasin D prevents this attachment. This is the first direct demonstration of the translocation of hexokinase to the plasma membrane in activated cells, and supports the view that enhanced hexokinase activity in the cortical region of the cytosol is an important early component of the macrophage activation process.