This study was undertaken to examine the effects of 12 weeks of skeletal unloading on parameters of calcium homeostasis, calcitropic hormones, bone histology, and biochemical markers of bone turnover in 11 normal subjects (9 men, 2 women; 34 ± 11 years of age). Following an ambulatory control evaluation, all subjects underwent 12 weeks of bed rest. An additional metabolic evaluation was performed after 12 days of reambulation. Bone mineral density declined at the spine (−2.9%, p = 0.092) and at the hip (−3.8%, p = 0.002 for the trochanter). Bed rest prompted a rapid, sustained, significant increase in urinary calcium and phosphorus as well as a significant increase in serum calcium. Urinary calcium increased from a pre-bed rest value of 5.3 mmol/day to values as high as 7.3 mmol/day during bed rest. Immunoreactive parathyroid hormone and serum 1,25-dihydroxyvitamin D declined significantly during bed rest, although the mean values remained within normal limits. Significant changes in bone histology included a suppression of osteoblastic surface for cancellous bone (3.1 ± 1.3% to 1.9 ± 1.5%, p = 0.0142) and increased bone resorption for both cancellous and cortical bone. Cortical eroded surface increased from 3.5 ± 1.1% to 7.3 ± 4.0% (p = 0.018) as did active osteoclastic surface (0.2 ± 0.3% to 0.7 ± 0.7%, p = 0.021). Cancellous eroded surface increased from 2.1 ± 1.1% to 4.7 ± 2.2% (p = 0.002), while mean active osteoclastic surface doubled (0.2 ± 0.2% to 0.4 ± 0.3%, p = 0.020). Serum biochemical markers of bone formation (osteocalcin, bone-specific alkaline phosphatase, and type I procollagen extension peptide) did not change significantly during bed rest. Urinary biochemical markers of bone resorption (hydroxyproline, deoxypyridinoline, and N-telopeptide of type I collagen) as well as a serum marker of bone resorption (type I collagen carboxytelopeptide) all demonstrated significant increases during bed rest which declined toward normal during reambulation. Thus, under the conditions of this study, the human skeleton appears to respond to unloading by a rapid and sustained increase in bone resorption and a more subtle decrease in bone formation.