The aim of this study was to determine cross-bridge number and kinetics in the diaphragm during fatigue and early recovery. Experiments were conducted in isolated mouse diaphragm (n=10). The force of a single cross-bridge (pi), the number of cross-bridges (m x 10(9) x mm2), the time cycle (tc) and the rate constants for cross-bridge attachment (f1) and detachment (g2) were calculated from the equations of A.F. Huxley. Following the fatigue protocol, peak isometric tension (Po) and maximum unloaded shortening velocity fell by 40+/-1% and 17+/-2%, respectively. In fatigued diaphragm, m fell by approximately 40% and returned to baseline after 10 min. When compared to baseline, g2 fell in fatigued diaphragm and remained significantly lower during the 15-min recovery period. In contrast, fatigue did not significantly modify pi, f1, or tc. There was a strong linear relationship between Po and m (p<0.001, r=0.988). No relationship was observed between tc and g2. These results indicate that changes in tension during fatigue and recovery run parallel to changes in the number of active cross-bridges, with no change in the force generated per cross-bridge. It is conceivable that fatigue durably impairs adenosine diphosphate release from the actomyosin complex without modifying the total duration of the cross-bridge cycle.