Dynamic weight bearing is important for normal growth and maintenance of the skeleton in humans and laboratory animals. Transforming growth factor-beta (TGF-β) has been implicated as having autocrine and paracrine actions in bone. The purpose of this study was to examine mRNA levels of TGF-β in skeletal tissues of growing male rats following skeletal unweighting during an 11-day spaceflight. Animals were sacrificed 5–8 h after the skeleton was reloaded. Spaceflight resulted in decreases in cortical bone area and periosteal bone formation, but no change in medullary area and endocortical bone formation. In addition, spaceflight had no effect on longitudinal bone growth. TGF-β was reduced relative to the ground controls in the hindlimb periosteum, but was not significantly altered in the growth zone of the tibial metaphysis. Similarly, mRNA levels for type I collagen were reduced in the periosteum, but not in the metaphysis of flight animals. The results suggest a potential role of TGF-β as an intermediate in the signal transduction pathway for mechanical loading. Further, they indicate skeletal tissue compartment-dependent changes in mRNA levels for TGF-β following weightlessness.