To study the effects of estrogen withdrawal on osteoclast number and osteoclast activity in the rat ovariectomy (OVX) model. We first cultured human CD34+ osteoclast precursor cells on bovine bone slices, allowing them to differentiate into mature resorbing osteoclasts. Secreted tartrate-resistant acid phosphatase 5b (TRACP 5b) and C-terminal cross-linked telopeptides of type I collagen (CTX) were determined from the culture medium. TRACP 5b correlated strongly with osteoclast number and CTX with osteoclast activity, facilitating their subsequent use in the rat OVX model. An 8 week OVX study was then performed including sham-operated rats receiving vehicle, OVX rats receiving vehicle, and OVX rats receiving 10 microg/kg/day 17 beta-estradiol (E2). Trabecular bone parameters were determined from the tibial metaphysis using peripheral quantitative computed tomography and histomorphometry. Osteoclast number was normalized with bone perimeter (N.Oc/B.Pm) and tissue area (N.Oc/T.Ar, indicating absolute number of osteoclasts). TRACP 5b and CTX were determined from fasting serum samples. Trabecular bone parameters indicated substantial bone loss after OVX that was prevented by E2. N.Oc/B.Pm increased after OVX, while N.Oc/T.Ar and TRACP 5b decreased, and TRACP 5b correlated strongly with N.Oc/T.Ar. However, CTX values increased after OVX, and the "resorption index" CTX/TRACP 5b showed more substantial changes than either CTX or TRACP 5b alone. These results show that TRACP 5b is a reliable marker of osteoclast number, and the index CTX/TRACP 5b is a useful parameter in rat OVX model. The high elevation of CTX/TRACP 5b values by OVX demonstrates that estrogen withdrawal generates high activity of osteoclasts in the rat OVX model.