Human osteosarcoma cells resistant to detachment by an Arg-Gly-Asp-containing peptide overproduce the fibronectin receptor. MG-63 human osteosarcoma cells were selected for attachment and growth in the presence of increasing concentrations of a synthetic peptide containing the cell attachment-promoting Arg-Gly-Asp sequence derived from the cell-binding region of fibronectin. Cells capable of attachment and growth in 5-mM concentrations of a peptide having the sequence Gly-Arg-Gly-Asp-Ser-Pro overproduce the cell surface receptor for fibronectin. In contrast, these cells show no differences in the numbers of vitronectin receptor they express as compared with the parental MG-63 cells. In agreement with the resistance of the selected cells to detachment by the peptide, 25-fold more Arg-Gly-Asp-containing peptide is required to prevent the attachment of these cells to fibronectin-coated surfaces than is needed to inhibit the attachment of MG-63 cells to the same substrate. However, similar concentrations of this peptide inhibit attachment of both cell lines to vitronectin-coated surfaces. The increase in fibronectin receptor is due to an increase in the levels of mRNA encoding the fibronectin receptor. Because of the nature of the selection process, we reasoned that this increase might be due to amplification of the fibronectin receptor gene, but no increase in gene copy number was detected by Southern blot analysis. The peptide-resistant cells display a very different morphology from that of the MG-63 cells, one that has a greater resemblance to that of osteocytes. The resistant cells also grow much more slowly than the MG-63 cells. The increased fibronectin receptor and altered morphology and growth properties were stable for at least 3 mo in the absence of peptide. The enhanced expression of the fibronectin receptor on the resistant cells indicates that cells are capable of altering the amount of fibronectin receptor on their surface in response to environmental factors and that this may in turn affect the phenotypic properties of the cell.