The solid‐phase synthesis of two porcine motilin derivatives, specifically biotinylated on the side chain of Lys20), was accomplished by preactivation of the protected amino acidsNx‐(9‐fluorenylmethoxycarbonyl)‐Nε‐biotinyl‐L‐lysine andNx‐(9‐fluorenylmethoxycarbonyl)‐Nε‐[N‐(biotinyl)‐6‐aminohexanoyl]‐L‐lysine with BOP/ HOBt/DIEA (1:1:2.5) followed by coupling to the support‐bound peptide substrate. The biotin moiety was stable to TFA cleavage and repetitive cycles of acylation, as evidenced by the high level of purity (>80%) of the crude peptides. This direct synthetic approach complements existing orthogonal protection strategies for the site‐specific biotinylation of peptides. The derivatized peptides were purified by RP‐HPLC and characterized by mass spectral and amino acid analysis. In binding studies using a rabbit antral smooth muscle homogenate, both [Leu13, Lys20(Nε‐biotinyl)]porcine motilin (3) and [Leu13, Lys20(Nε‐[N‐(biotinyl)‐6‐aminohexanoyl])]porcine motilin (4) possessed nearly equal affinities for the motilin receptor (IC50= 0.89 and 1.2 nM, respectively) as native porcine motilin (1) (IC50= 0.76 nM). The biotinylated peptides were also highly potent in tissue bath assays employing rabbit duodenal smooth muscle segments. In contrast, commercially available [Nx‐biotinylPhe1]porcine motilin (5) had markedly lower affinity in the binding assay (IC50= 30 nM). The relative bioactivities of these receptor probes are in accord with previous synthetic studies on motilin which demonstrated the importance of the amino‐terminal segment in the high affinity interaction between the peptide and its receptor. Analog 3 retained high affinity for the motilin receptor in the presence of avidin. Therefore, this peptide is expected to be a valuable tool for the isolation and