The work of breathing (W(B)), and thus the energy requirement of the respiratory muscles, is increased any time minute ventilation (VE) is elevated, by either exercise or voluntary hyperventilation. Respiratory muscle O2 consumption (VRM(O2)) in humans has generally been estimated by having subjects breathe at a level comparable to that during exercise while the change in O2 consumption (VO2) is measured. The difference between VO2 at rest and during hyperventilation is attributed to the respiratory muscles and is assumed to be similar to VRM(O2) during exercise at the same VE. However, it has been suggested that W(B) differs between exercise and hyperventilation and that W(B) during exercise is lower than during hyperventilation at the same VE. In this study we measured W(B) during exercise and hyperventilation and from these measurements estimated VRM(O2). W(B), VE, and VO2 were measured in five male subjects during rest and during exercise or hyperventilation at levels of VE ranging from 30 to 130 I/min. VE/W(B) relationship was determined for both hyperventilation and exercise. Multiple regression analysis showed that the shape of the two curves was different (P < 0.0001), with W(B) at high levels of VE being less-than-or-equal-to 25% higher in hyperventilation than in exercise. In a second study in which frequency, tidal volume, and duty cycle were controlled as well as VE, there was. no difference in W(B) between exercise and hyperventilation. VO2 was significantly correlated with W(B), and the estimated VRM(O2) did not increase as a fraction of total VO2 as exercise intensity rose. From these results we suggest that when carefully controlled for both pattern and VE, hyperventilation can be used to mimic exercise and to estimate the metabolic cost of breathing. However, if only VE is controlled, it is necessary to measure W(B) to estimate the energy used by the respiratory muscles.