This study compared in vivo measurements of muscle metabolism in humans with magnetic resonance spectroscopy (MRS) and in vitro analysis of biopsies. Healthy subjects [4 young males, 28.2 +/- 6.8 (SD) yr, and 6 older subjects (5 males, 1 female), 66 +/- 6.0 yr] performed a maximal cycle ergometer test, and MRS measurements of the calf muscles and needle biopsies of the lateral gastrocnemius were performed. Biopsies were analyzed for fiber type and citrate synthase (CS) activity. MRS measurements of inorganic phosphate (Pi), phosphocreatine (PCr), ATP, and pH were made using a 1.8-T 78-cm clear-bore magnet-and-spectrometer system. Two or three 5-min bouts of plantar flexion were performed against variable resistance to deplete PCr levels to 50% of resting values (mean end pH 6.99). PCr values during recovery were fit to an exponential curve, and the rate constant (PCrrate) was calculated. PCrrate was used as an index of oxidative metabolism. Older subjects had lower peak O2 uptake (VO2 peak) values (19.2 +/- 5.6 vs. 49.5 +/- 8.1 ml O2.min-1 x kg-1), CS activities (16 +/- 2.8 vs. 25 +/- 2.6 mmol.kg wet wt-1 x min-1), and PCrrate values (25.3 +/- 8. vs. 37.5 +/- 5.3 mmol PCr.kg wet wt-1.min-1) than young subjects. PCrrate correlated with CS activity, and both PCrrate and CS activity correlated with VO2 peak (P < 0.05). No correlations were found between percent fiber type and PCrrate, CS activity, and VO2 peak. These results support studies that showed decreases in muscle metabolism with age in healthy humans and show a good correlation between in vivo and in vitro measurements of oxidative metabolism.