Green fluorescent protein (GFP)-expressing transgenic mice were produced containing a 3.6-kilobase (kb; pOBCol3.6GFPtpz) and a 2.3-kb (pOBCol2.3GFPemd) rat type I collagen (Col1a1) promoter fragment. The 3.6-kb promoter directed strong expression of GFP messenger RNA (mRNA) to bone and isolated tail tendon and lower expression in nonosseous tissues. The 2.3-kb promoter expressed the GFP mRNA in the bone and tail tendon with no detectable mRNA elsewhere. The pattern of fluorescence was evaluated in differentiating calvarial cell (mouse calvarial osteoblast cell [mCOB]) and in marrow stromal cell (MSC) cultures derived from the transgenic mice. The pOBCol3.6GFPtpz-positive cells first appeared in spindle-shaped cells before nodule formation and continued to show a strong signal in cells associated with bone nodules. pOBCol2.3GFPemd fluorescence first appeared in nodules undergoing mineralization. Histological analysis showed weaker pOBCol3.6GFPtpz-positive fibroblastic cells in the periosteal layer and strongly positive osteoblastic cells lining endosteal and trabecular surfaces. In contrast, a pOBCol2.3GFPemd signal was limited to osteoblasts and osteocytes without detectable signal in periosteal fibroblasts. These findings suggest that Col1a1GFP transgenes are marking different subpopulations of cells during differentiation of skeletal osteoprogenitors. With the use of other promoters and color isomers of GFP, it should be possible to develop experimental protocols that can reflect the heterogeneity of cell differentiation in intact bone. In primary culture, this approach will afford isolation of subpopulations of these cells for molecular and cellular analysis.