The osteoprotegerin (OPG)/receptor activator of NF-kappaB ligand (RANKL) axis is thought to be involved in the upregulation of bone turnover following sex steroid deficiency. Here, we investigated the effects of orchiectomy (ORX) on bone turnover and free soluble RANKL (sRANKL) in aged rats. Free, bioactive sRANKL is a critical determinant and key mediator for survival and activity of mature osteoclasts. Thirty-three 9-month-old male Fischer-344 rats were either ORX or sham-operated (SHAM). Following in vivo fluorochrome labeling, vehicle (ricinus oil/benzyl benzoate)-treated SHAM and vehicle- or testosterone undecanoate (T, 6 mg/kg s.c. once weekly)-treated ORX rats (n=8-9 each) were killed 2 months after surgery. Vehicle-treated ORX rats showed lower seminal vesicle weight, loss of proximal tibial trabecular bone mineral density, and reduced cortical thickness at the tibial shaft as measured by peripheral quantitative computed tomography relative to SHAM controls. Bone loss in vehicle-treated ORX rats was associated with enhanced bone turnover as evidenced by increases in tibial cancellous bone formation rate, osteoclast numbers, urinary excretion of calcium and deoxypyridinoline, and serum osteocalcin. T treatment of ORX rats restored seminal vesicle weight to SHAM control levels, and completely protected against post-ORX bone loss by suppressing bone turnover. Free sRANKL concentrations in bone marrow supernatants harvested from the proximal femur were about 3-fold higher in vehicle-treated ORX relative to SHAM rats, and returned to SHAM control levels in T-treated ORX rats. mRNA abundance of matrix metalloproteinase-14 (MMP-14) in bone marrow was 4-fold higher in vehicle-treated ORX rats relative to SHAM rats. T treatment of ORX rats suppressed MMP-14 mRNA expression to SHAM control levels. We conclude that orchiectomy increases the concentration of free sRANKL in bone marrow of aged rats. In addition, increased shedding of membrane-bound RANKL by MMP-14 may be a pivotal mechanism resulting in augmented free sRANKL concentrations in the bone marrow environment after androgen withdrawal.