The mechanism of the loss of skeletal muscle mass that occurs during spaceflight is not well understood. Myostatin has been proposed as a negative modulator of muscle mass, and IGF-I and IGF-II are known positive regulators of muscle differentiation and growth. We investigated whether muscle loss associated with spaceflight is accompanied by increased levels of myostatin and a reduction in IGF-I and -II levels in the muscle, and whether these changes correlate with an increase in muscle proteolysis and apoptosis. Twelve male adult rats sent on the 17-day NASA STS-90 NeuroLab space flight were divided upon return to earth into two groups, and killed either 1 day later (R1) or after 13 days of acclimatization (R13). Ground-based control rats were maintained for the same periods in either vivarium (R3 and R15, respectively), or flight-simulated cages (R5 and R17, respectively). RNA and protein were isolated from the tibialis anterior, biceps femoris, quadriceps, and gastrocnemius muscles. Myostatin, IGF-I, IGF-II and proteasome 2c mRNA concentrations were determined by reverse transcription/PCR; myostatin and ubiquitin mRNA were also measured by Northern blot analysis; myostatin protein was estimated by immunohistochemistry; the apoptotic index and the release of 3-methylhistidine were determined respectively by the TUNEL assay and by HPLC. Muscle weights were 19-24% lower in the R1 rats compared with the control R3 and R5 rats, but were not significantly different after the recovery period. The myostatin/beta-actin mRNA ratios (means+/-s.e.m. ) were higher in the muscles of the R1 rats compared with the control R5 rats: 5.0-fold in tibialis (5.35 +/- 1.85 vs 1.07 +/- 0.26), 3.0-fold in biceps (2.46+/-0.70 vs 0.81 +/- 0.04), 1.9-fold in quadriceps (7.84 +/- 1.73 vs 4.08 +/- 0.52), and 2.2-fold in gastrocnemius (0.99 +/- 0.35 vs 0.44 +/- 0.17). These values also normalized upon acclimatization. Our antibody against a myostatin peptide was validated by detection of the recombinant human myostatin protein on Western blots, which also showed that myostatin immunostaining was increased in muscle sections from R1 rats, compared with control R3 rats, and normalized upon acclimatization. In contrast, IGF-II mRNA concentrations in the muscles from R1 rats were 64-89% lower than those in R3 animals. With the exception of the gastrocnemius, IGF-II was also decreased in R5 animals maintained in flight-simulated cages, and normalized upon acclimatization. The intramuscular IGF-I mRNA levels were not significantly different between the spaceflight rats and the controls. No increase was found in the proteolysis markers 3-methyl histidine, ubiquitin mRNA, and proteasome 2C mRNA. In conclusion, the loss of skeletal muscle mass that occurs during spaceflight is associated with increased myostatin mRNA and protein levels in the skeletal muscle, and a decrease in IGF-II mRNA levels. These alterations are normalized upon restoration of normal gravity and caging conditions. These data suggest that reciprocal changes in the expression of myostatin and IGF-II may contribute to the multifactorial pathophysiology of muscle atrophy that occurs during spaceflight.