The design of a fibronectin-mimetic peptide that specifically binds to the alpha(5)beta(1) integrin has been widely studied due to this integrin's participation in many physiological and pathological processes. A promising design for such a peptide includes both the primary binding site RGD and the synergy site PHSRN connected by a linker and extended off a surface by a spacer. These four building blocks were used for the headgroup of a peptide-amphiphile that we designed previously, which was shown, for the first time, to provide comparable cell adhesion levels to native fibronectin. This peptide-amphiphile, PR_b, employed a hydrophobic tail that had two C16 dialkyl tails, a glutamic acid tail linker, a -(CH2)2- tail spacer, and was connected to the N-terminus of a peptide composed of a headgroup spacer, the synergy site sequence, a headgroup linker mimicking both the distance and hydrophobicity/hydrophilicity present in the native protein, and finally the primary binding sequence. To further study PR_b's success, different peptide-amphiphiles were designed that systematically studied the effect of the peptide headgroup linker length and hydrophobicity/hydrophilicity and the effect of the headgroup spacer length on human umbilical vein endothelial cell adhesion and fibronectin production. In addition to evaluating the performance of different peptide sequences, this work also outlines a logical approach that can be applied to the future design of any protein-mimetic peptide that combines two active binding sites.