Concentrations of prostaglandin E2, prostaglandin F2 alpha (PGF2 alpha), and its metabolite 13,14-dihydro-15-keto prostaglandin F2 alpha (DHKF2 alpha) were measured in human milk at three different stages of lactation in mothers of premature and term infants. Before radioimmunoassay, our samples were purified by solid-phase extraction with C18Sep-Pak cartridges and reverse-phase high-performance liquid chromatography. When concentrations of the three prostaglandins were compared in the milk of mothers delivering prematurely and at term, very low concentrations were measured and no differences were observed. Concentrations of prostaglandins also remained essentially the same at the various stages of lactation. However, the mean ratio of DHKF2 alpha to PGF2 alpha was higher in samples from mothers who delivered early than in samples from those who delivered at term. These ratios were comparable to those reported in the literature for human plasma. The functional significance of these compounds in human milk remains undetermined.