The objective of this study was to determine the effects of spaceflight on the structure of the tendon-bone junction (TBJ). Pregnant rats either flew in the space shuttle Atlantis (flight group; F) or were exposed to simulated launch and landing protocols (synchronous control group; SC) during days 9-19 of pregnancy. Following birth of their pups, maternal hindlimbs were studied using scanning electron and light microscopic histomophometric techniques. The tibial and calcaneal tuberosities, the fibular head, and the tibia-fibula junction were studied. Myofiber density and cross-sectional area of the quadratus femoris and soleus muscles and diameters of the calcaneal and patellar tendons were also evaluated. Cortical erosion was significantly greater at the tibial tuberosity and the fibular head in F animals compared to SC animals (P < 0.001). Sharpey fiber density was significantly less at the tibial tuberosity and fibular head in F animals compared to SC animals (P < 0.001). The myofiber area of both the soleus and quadratus femoris muscles and the diameters of both calcaneal and patellar tendons were significantly less in F compared to SC rats (P < 0.05). Our data illustrate that the TBJ morphology is affected by spaceflight at the attachment sites of the soleus and quadratus femoris muscles in pregnant animals, which could adversely affect their physical properties. These atrophic TBJ changes could have resulted from atrophy of the adjacent muscles and their tendons. Atrophic changes in the structure of the TBJ could predispose an animal to injury following spaceflight, when normal gravity conditions are reestablished.