An acceleration of ATP synthesis by anaerobic glycolysis provides important compensation for interference with respiration in a variety of cells. Effective compensation for an inhibition of respiration, however, can occur in cells in which glucose entry is rate limiting only if sufficient glucose becomes available through an enhancement of transport. We present here a detailed study of the effects of inhibition of respiration in Clone 9 cells, a continuous cell line characterized by low internal glucose concentrations (less than 10% that of the external medium) and minimal stores of glycogen. Exposure of these cells to 5 mM cyanide results in a 90% fall in cell ATP and a twofold rise in cell Na+ within 20 min. By the end of 1 h, however, there is a 4.5- to 7-fold increase in cytochalasin B-inhibitable glucose transport that is accompanied by a parallel increase in the rate of lactate production, a partial recovery of cell ATP, and no further rise in cell Na+. The acute fall in ATP resulting from a submaximally effective concentration of cyanide (0.5 mM) is moreover followed by a time-dependent recovery of cell ATP to near-normal levels and subsequent resistance to challenge with even 5 mM cyanide. The stimulation of facilitative glucose transport resulting from exposure to cyanide is attributable to an increase in maximal velocity rather than to a change in Km and persists for more than 2 h after removal of the inhibitor. These results demonstrate that, in these cells characterized by low internal glucose concentrations, regulation of glucose entry is of central importance in ATP homeostasis and that a major component of the adaptive response to an inhibition of respiration is a time-dependent increase in glucose transport.