The distribution of two integrins, the fibronectin receptor and the vitronectin receptor, has been explored in an endothelium-derived cell line plated onto various substrata. On a fibronectin substratum, in the presence of serum, these cells develop focal contacts that contain the fibronectin receptor, whereas the vitronectin receptor is diffusely distributed over the cell surface. Conversely, cells plated onto vitronectin-coated coverslips concentrate only the vitronectin receptor within focal contacts. The accumulation of the vitronectin receptor within focal contacts also occurs when the cells are plated on uncoated coverslips but in the presence of serum. Therefore, we conclude that under normal culture conditions (i.e. in serum-containing media), the vitronectin receptor is the predominant form of integrin involved in substratum adhesion. This conclusion is supported by experiments in which cells were cultured on fibronectin-coated coverslips in the presence of serum. Initially these cells developed focal contacts containing only the fibronectin receptor. Within several hours, however, there was a progressive replacement of focal contacts containing the fibronectin receptor by focal contacts expressing the vitronectin receptor. After approximately 12 h in culture, most cells contained focal contacts expressing only the vitronectin receptor. Focal contacts containing either the fibronectin or vitronectin receptor were both associated with the termini of stress fibres and contained the proteins talin and vinculin. These observations lead us to propose that the cell does not discriminate between these different integrins when assembling the cytoskeletal components at the cytoplasmic face of focal contacts.