Experimental conditions and parameters involved in high performance liquid chromatography (HPLC) separations of the peptide hormone oxytocin and seven of its diastereoisomers, namely [1‐hemi‐d‐cystine]‐, [2‐d‐tyrosine]‐, [4‐d‐glutamine]‐, [5‐d‐asparagine]‐, [6‐hemi‐d‐cystine‐], [7‐d‐proline]‐, and [8‐d‐leucine]‐oxytocin, on reverse phase columns were investigated. The effects of solvent, pH, and salt concentration were studied. Using the solvent systems 10% tetrahydrofuran‐ammonium acetate buffer or 18% acetonitrile‐ammonium acetate buffer and the üBondapak C18support, oxytocin was separated from each of its diastereoisomers under all conditions studied, but the order of elution of diastereoisomers was highly dependent on solvent and to a lesser extent on pH. Separations of the hormone and its diastereoisomers on reverse phase HPLC and on classical partition chromatography on Sephadex G‐25 were compared. The results are discussed in terms of the interactions of the solute with the reverse phase column and the solvent system. Implications of these findings in terms of the different