Prolonged mechanical ventilation results in diaphragmatic atrophy and contractile dysfunction in animals. We hypothesized that mechanical ventilation-induced diaphragmatic atrophy is associated with decreased synthesis of both mixed muscle protein and myosin heavy chain protein in the diaphragm. To test this postulate, adult rats were mechanically ventilated for 6, 12, or 18 hours and diaphragmatic protein synthesis was measured in vivo. Six hours of mechanical ventilation resulted in a 30% decrease (p < 0.05) in the rate of mixed muscle protein synthesis and a 65% decrease (p < 0.05) in the rate of myosin heavy chain protein synthesis; this depression in diaphragmatic protein synthesis persisted throughout 18 hours of mechanical ventilation. Real-time polymerase chain reaction analyses revealed that mechanical ventilation, in comparison with time-matched controls, did not alter diaphragmatic levels of Type I and IIx myosin heavy chain messenger ribonucleic acid levels in the diaphragm. These data support the hypothesis that mechanical ventilation results in a decrease in both mixed muscle protein and myosin heavy chain protein synthesis in the diaphragm. Further, the decline in myosin heavy chain protein synthesis does not appear to be associated with a decrease in myosin heavy chain messenger ribonucleic acid.