Intermittent administration of parathyroid hormone (PTH) increases bone mass, at least in part, by increasing the number of osteoblasts. One possible source of osteoblasts might be conversion of inactive lining cells to osteoblasts, and indirect evidence is consistent with this hypothesis. To better understand the possible effect of PTH on lining cell activation, a lineage tracing study was conducted using an inducible gene system. Dmp1-CreERt2 mice were crossed with ROSA26R reporter mice to render targeted mature osteoblasts and their descendents, lining cells and osteocytes, detectable by 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) staining. Dmp1-CreERt2(+):ROSA26R mice were injected with 0.25 mg 4-OH-tamoxifen (4-OHTam) on postnatal days 3, 5, 7, 14, and 21. The animals were euthanized on postnatal day 23, 33, or 43 (2, 12, or 22 days after the last 4-OHTam injection). On day 43, mice were challenged with a subcutaneous injection of human PTH (1-34, 80 µg/kg) or vehicle once daily for 3 days. By 22 days after the last 4-OHTam injection, most X-gal (+) cells on the periosteal surfaces of the calvaria and the tibia were flat. Moreover, bone formation rate and collagen I(α1) mRNA expression were decreased at day 43 compared to day 23. After 3 days of PTH injections, the thickness of X-gal (+) cells increased, as did their expression of osteocalcin and collagen I(α1) mRNA. Electron microscopy revealed X-gal-associated chromogen particles in thin cells prior to PTH administration and in cuboidal cells following PTH administration. These data support the hypothesis that intermittent PTH treatment can increase osteoblast number by converting lining cells to mature osteoblasts in vivo.