Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we have developed a simple method to isolate myosin heavy chain (MHC) and actin from small (60-80 mg) human skeletal muscle samples for the determination of their fractional synthesis rates. The amounts of MHC and actin isolated are adequate for the quantification of [13C]leucine abundance by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Fractional synthesis rates of mixed muscle protein (MMP), MHC, and actin were determined in six healthy young subjects (27 +/- 1 yr) after they received a 14-h intravenous infusion (prime = 7.58 micromol/kg body wt, constant infusion = 7.58 micromol. kg body wt-1. h-1) of [1-13C]leucine. The fractional synthesis rates of MMP, MHC, and actin were found to be 0.0468 +/- 0.0048, 0.0376 +/- 0. 0033, and 0.0754 +/- 0.0078%/h, respectively. Overall, the synthesis rate of MHC was 20% lower (P = 0.012), and the synthesis rate of actin was 61% higher (P = 0.060, not significant) than the MMP synthesis rate. The isolation of these proteins for isotope abundance analysis by GC-C-IRMS provides important information about the synthesis rates of these specific contractile proteins, as opposed to the more general information provided by the determination of MMP synthesis rates.