The cell-substrate interface plays a key role in the regulation of cell behaviour. Defining the properties of this interface is particularly important for human embryonic stem (hES) cell culture, because changes in this environment can regulate hES cell differentiation. It has been established that fibronectin-coated surfaces can promote the attachment, growth and maintenance of the undifferentiated phenotype of hES cells. We investigated the influence of the surface density of adsorbed fibronectin on hES cell behaviour in defined serum-free culture conditions and demonstrated that only 25 per cent surface saturation was required to maintain attachment, growth and maintenance of the undifferentiated phenotype. The influence of surface-adsorbed fibronectin fragments was compared with whole fibronectin, and it was demonstrated that the 120 kDa fragment central binding domain alone was able to sustain hES cells in an undifferentiated phenotype in a similar fashion to fibronectin. Furthermore, hES cell attachment to both fibronectin and the 120 kDa fragment was mediated by integrin α5β1. However, although a substrate-attached synthetic arginine-glycine-aspartic acid (RGD) peptide alone was able to promote the attachment and spreading of fibroblasts, it was inactive for hES cells, indicating that stem cells have different requirements in order to attach and spread on the central fibronectin RGD-cell-binding domain. This study provides further information on the characteristics of the cell-substrate interface required to control hES cell behaviour in clearly defined serum-free conditions, which are needed for the development of therapeutic applications of hES cells.