Collagen synthesis and tissue remodeling are involved in many diseases; therefore, collagen-specific binding agents have been developed to study collagen changes in various tissues. Based on a recently reported collagen binding peptide, which contains unnatural biphenylalanine (Bip) amino acid residue, constructs with various structure variations were synthesized to explore the contributions of unnatural Bip residue, conformational restrain, and amino acid sequence in collagen recognition. Their binding efficiency to collagens was evaluated in vitro using pure collagens. The results indicate that the C-terminal unnatural Bip residue, rather than the peptide sequence or conformational restrain, dominated the collagen I binding. Subsequent tissue binding study showed that the selected peptide did not offer preferential selectivity over collagen I in tissue, suggesting that a simple in vitro binding assay cannot adequately model the complex biological environment. © 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 167–173, 2013.