It has been reported that oxygen uptake (VO₂) increases exponentially with levels of the pedal rate during cycling. The purpose of this study was therefore to test the hypothesis that the O₂ cost for internal power output (P_int) exerted in exercising muscle itself would be larger than for an external power output (P_ext) calculated from external load and pedal rate during cycling exercise under various conditions of P_int and P_ext in a large range of pedal rates. The O₂ cost (ΔVO₂/ Δpower output) was investigated in three experiments that featured different conditions on a cycle ergometer that were carried out at the same levels of total power output (P_tot; sum of P_int and P_ext) (Exp. 1), P_ext (Exp. 2) and load (Exp. 3). Each experiment consisted of three exercise tests with three levels of pedal rate (40 rpm for a lower pedal rate: LP; 70–80 rpm for a moderate pedal rate: MP; and 100–120 rpm for a higher pedal rate: HP) lasting for 2–3 min of unloaded cycling followed by 4–5 min of loaded cycling. Blood lactate accumulations (2.3–3.4 mmol l⁻¹) at the HP were significantly higher compared with the LP (0.6–0.9 mmol l⁻¹) and MP (0.9–1.0 mmol l⁻¹) except for the LP in Exp. 1. The VO₂ (360–432 ml min⁻¹ for LP, 479–644 ml min⁻¹ for MP, 960–1602 ml min⁻¹ for HP) during unloaded cycling in the three experiments increased exponentially with increasing pedal rates regardless of P_ext=0. Moreover, the slope of the VO₂-P_int (13.7 ml min⁻¹ W⁻¹) relation revealed a steeper inclination than that of the VO₂-P_ext (10.2 ml min⁻¹ W⁻¹) relation. We concluded that the O₂ cost for P_int was larger than for P_ext during the cycling exercises, indicating that the O₂ cost for P_tot could be affected by the ratio of P_int to P_tot due to the levels of pedal rate.