The incorporation of homoserine [Hse] into synthetic peptides has proved extremely difficult due to the ease with which homoserine derivatives form a γ-lactone. We now wish to report the synthesis of [4-homoserine]oxytocin. We wished to obtain this peptide to test our earlier hypothesis as to why threonine substitution in the 4 position in oxytocin enhanced both oxytocic activity (O) and oxytocic-antidiuretic (O/A) selectivity relative to oxytocin. The key to this synthesis was the preparation of Nα-tert-butyloxycarbonyl-O-benzyl-L-homoserine [Boc-Hse(Bzl)] by a modification of a recently published method used for the benzylation of Nα-tert-butyloxycarbonylserine (Boc-Ser). Boc-Hse(Bzl) was incorporated into the protected nonapeptide Z-Cys(Bzl)-Tyr(Bzl)-Ile-Hse(Bzl)-Asn-Cys(Bzl)-Pro-Leu-Gly-NH₂ using the solid-phase method by procedures previously described from these laboratories. Following deblocking, cyclization, and purification in the usual manner the desired peptide, [4-homoserine]oxytocin, was obtained. An examination of its pharmacological properties provided the following potencies (units/mg): rat oxytocic (O) 125 ± 13; rat antidiuretic (A) 0.24 ± 0.03. Its oxytocic-antidiuretic ratio is thus 521. These data indicate that threonine and homoserine when substituted at position 4 in oxytocin exert similar effects on O/A selectivity (cf. [4-threonine]oxytocin has an O/A ratio of 512) but that threonine exerts a unique effect in leading to enhanced oxytocic activity (cf. [4-threonine]oxytocin has an oxytocic potency of 923 ± 96 units/mg). This successful synthesis of Boc-Hse(Bzl) and its incorporation into [4-homoserine]oxytocin now make possible the synthesis of homoserine analogues of other peptides.