The luteal phase of the female menstrual cycle is associated with both 1) elevated serum progesterone (P4) and estradiol (E2), and 2) reduced insulin sensitivity. Recently, we demonstrated a link between skeletal muscle mitochondrial H(2)O(2) emission (mE(H2O2)) and insulin resistance. To determine whether serum levels of P4 and/or E(2) are related to mitochondrial function, mE(H2O2) and respiratory O(2) flux (Jo(2)) were measured in permeabilized myofibers from insulin-sensitive (IS, n = 24) and -resistant (IR, n = 8) nonmenopausal women (IR = HOMA-IR > 3.6). Succinate-supported mE(H2O2) was more than 50% greater in the IR vs. IS women (P < 0.05). Interestingly, serum P4 correlated positively with succinate-supported mE(H2O2) (r = 0. 53, P < 0.01). To determine whether P4 or E2 directly affect mitochondrial function, saponin-permeabilized vastus lateralis myofibers biopsied from five nonmenopausal women in the early follicular phase were incubated in P4 (60 nM), E2 (1.4 nM), or both. P4 alone inhibited state 3 Jo(2), supported by multisubstrate combination (P < 0.01). However, E2 alone or in combination with P4 had no effect on Jo(2). In contrast, during state 4 respiration, supported by succinate and glycerophosphate, mE(H2O2) was increased with P4 alone or in combination with E2 (P < 0.01). The results suggest that 1) P4 increases mE(H2O2) with or without E2; 2) P4 alone inhibits Jo(2) but not when E2 is present; and 3) P4 is related to the mE(H2O2) previously linked to skeletal muscle insulin resistance.