Either increased protein synthesis or prolonged protein half-life is necessary to support the excessive muscle growth and maintenance of enlarged muscles in myostatin-deficient mice. This issue was addressed by determining in vivo rates of myofibrillar protein synthesis in mice with constitutive myostatin deficiency (Mstn^E3/E3) or normal myostatin expression(Mstn^+/+), by measuring tracer incorporation after a systemic flooding dose of ring-²H₅-L-phenylalanine. At 5-6 weeks of age, Mstn^E3/E3 mice had increased muscle mass (40%), fractional rates of myofibrillar synthesis (14%), and protein synthesis per whole muscle (60%) relative to Mstn^+/+ mice. With maturation, fractional rates of synthesis declined > 50% in parallel with decreased DNA and RNA (total, 28S rRNA, and polyA RNA) concentrations in muscle. At 6 months of age, Mstn^E3/E3mice had even greater increases in muscle mass (90%) and myofibrillar synthesis per muscle (85%) relative to Mstn^+/+ mice, but the fractional rate of synthesis was normal. Estimated myofibrillar protein half-life was not affected by myostatin deficiency. Muscle DNA concentrations were reduced in both young and mature Mstn^E3/E3 mice while RNA concentrations were normal, so that the ratio of RNA to DNA was ~30% greater than normal in Mstn^E3/E3 mice. Thus, the increased protein synthesis and RNA content per muscle in myostatin-deficient mice cannot be explained entirely by an increased number of myonuclei.