Synthetic peptides derived from the cell-binding domain of fibronectin have previously been found to inhibit fibronectin-mediated adhesion in vitro competitively and reversibly, as well as inhibiting cell migratory events in vivo. The amino acid sequence specificity required for this inhibitory activity has been examined further using variations of the originally identified active peptide sequences. The most active small peptide was found to be the pentapeptide Gly-Arg-Gly-Asp-Ser. Although the tetrapeptide Arg-Gly-Asp-Ser was found to retain substantial activity, it was approximately threefold less active. An "inverted" peptide sequence with these same four amino acids arranged in the mirror symmetrical sequence Ser-Asp-Gly-Arg was found to be nearly as active as the forward sequence. However, the same inverted tetrapeptide sequence embedded in a synthetic decapeptide derived from a sequence of histocompatibility antigens has minimal activity, suggesting the importance of adjacent sequences in modifying the activity of such peptides. Neither substitution of amino acids of the same charge nor reversal of the positions of the two charged amino acids retains biological activity. Decreasing the spacing between the charged residues also causes a loss of activity. Our results suggest the hypothesis that this adhesive recognition signal consists of a specific arrangement of one acidic and one basic charged group and additional information provided by adjacent amino acids.