Selected biochemical characteristics of diaphragm muscle were compared among several orders of adult mammals (cattle, swine, rabbit, guinea pig, rat, and mouse) with known differences in resting breathing frequencies (f, range = 15-138). Diaphragms from smaller animals had significantly higher citrate synthase (CS) and phosphofructokinase (PFK) activities and substrate oxidation rates than larger animals. Ranges of activities for CS and PFK were 93-27 and 58-39 mumol.g-1.min-1, respectively; and 34-5 and 19-2 nmol.g-1.min-1 for [U-14C]glucose (GLU) and [1-14C]palmitate (PAL) oxidation, respectively. The percent of native fast myosin (FM) isoforms was significantly different among groups. Mouse diaphragm had the highest % FM (88.6%), whereas the lowest values (7.5%) were observed in cattle diaphragm. Myosin ATPase (M-ATPase, pH 9.8) activity was significantly lower in cattle (0.06 mumol.mg protein-1.min-1) and swine (0.38 mumol.mg protein-1.min-1) diaphragm than in other mammals (range of 1.14-0.67 mumol.mg protein-1.min-1). Correlation coefficients determined among means of measured biochemical parameters and established values of f indicated that CS activity and substrate oxidation rates were significantly correlated with f (r = 0.92, 0.92, 0.86 for CS, GLU, PAL, respectively) and the % FM increased with f. M-ATPase (pH 9.8) was significantly correlated with % FM (r = 0.85), whereas PFK and M-ATPase activities were not closely associated with f. It was concluded that f in mammals is significantly correlated with the biochemical parameters of aerobic capacity and is associated with the percent of FM isoforms in the diaphragm.