The effects of mechanical loading on the osteoblast phenotype remain unclear because of many variables inherent to the current experimental models. This study reports on utilization of a mouse tooth movement model and a semiquantitative video image analysis of in situ hybridization to determine the effect of mechanical loading on cell-specific expression of type I collagen (collagen I) and alkaline phosphatase (ALP) genes in periodontal osteoblasts, using nonosseous cells as an internal standard. The histomorphometric analysis showed intense osteoid deposition after 3 days of treatment, confirming the osteoinductive nature of the mechanical signal. The results of in situ hybridization showed that in control periodontal sites both collagen I and ALP mRNAs were expressed uniformly across the periodontium. Treatment for 24 hours enhanced the ALP mRNA level about twofold over controls and maintained that level of stimulation after 6 days. In contrast, collagen I mRNA level was not affected after 24 hours of treatment, but it was stimulated 2.8-fold at day 6. This increase reflected enhanced gene expression in individual osteoblasts, since the increase in osteoblast number was small. These results indicate that (1) the mouse model and a semiquantitative video image analysis are suitable for detecting osteoblast-specific gene regulation by mechanical loading; (2) osteogenic mechanical stress induces deposition of bone matrix primarily by stimulating differentiation of osteoblasts, and, to a lesser extent, by an increase in number of these cells; (3) ALP is an early marker of mechanically-induced differentiation of osteoblasts. (4) osteogenic mechanical stimulation in vivo produces a cell-specific 2.8-fold increase in collagen gene expression in mature, matrix-depositing osteoblasts located on the bone surface and within the osteoid layer.