An understanding of differential gene expression in highly metastatic osteosarcoma could provide gene targets for treatment of metastases. We compared gene expression profiles of high- (LM7) and low- (LM2) metastatic SaOS2-derived cell lines in an in vitro tissue culture model and examined several differentially regulated genes in vivo in a murine orthotopic xenograft model. We hypothesized an orthotopic inoculation of LM2 and LM7 cells would establish a primary lesion and the gene expression profile of cells grafted in this fashion would resemble the gene expression profile observed in an in vitro model. Thirty-five days after inoculation, animals were euthanized and both tibiae were harvested and rapidly frozen in liquid nitrogen. Human-specific GAPDH mRNA was present in two of four tibias inoculated with LM2 cells and three of four tibias inoculated with LM7 cells. Tibiae displaying the presence of human cells were assayed by semiquantitative reverse transcriptase polymerase chain reaction. We observed poor correspondence of in vitro to in vivo gene expression for either cell line. Accordingly, in vitro osteosarcoma gene expression data must be interpreted with caution until confirmed in vivo. Our orthotopic injection model allowed in vivo study of differential gene expression between these two cell lines but did not show radiographic evidence of an established primary lesion.