In murine bone marrow-derived macrophages, lysosomes often form tubulovesicular compartments, whose extended distribution in the cytoplasm depends on the integrity of cytoplasmic microtubules. When macrophages with fluorescently labeled lysosomes were plated onto coverslips opsonized with IgG, they engaged that surface in a phagocytic response (frustrated phagocytosis). The tubular lysosomal compartment of these cells collected in a central, perinuclear region, despite the continued presence of a radiating array of cytoplasmic microtubules. Using methods developed in the study of melanophores, we permeabilized macrophages engaged in frustrated phagocytosis, then re-activated lysosome extension along microtubules. Permeabilization was selective for plasma membranes, in that high molecular weight probes such as trypan blue or IgG could enter cells, while fluorescent probes previously loaded into lysosomes via endocytosis remained contained therein. Addition of 2 mM ATP, GTP or UTP to these permeabilized cell models produced centrifugal extension of tubular lysosomes. Selective depletion of ATP, using Escherichia coli glycerol kinase, inhibited ATP-dependent extension but not that which occurred with GTP or UTP, indicating that the mechanism of radial movement can use any of these three nucleotide triphosphates. Extension was independent of pH between 6.8 and 7.4, and was inhibited by AMP-PNP and by GMP-PNP. Depolymerization of cytoplasmic microtubules with nocodazole prevented subsequent ATP-inducible lysosome extension, whereas preincubation of cells with cytochalasin D did not inhibit the response. These results are consistent with the in vitro mechanochemical properties of kinesin (Cohn et al., 1989), and support earlier evidence, obtained in living cells, that kinesin is the mechanochemical motor of lysosome extension along microtubules in macrophages.