The free and esterified (舠bound舡) hydroxy acids from butterfat were isolated by column‐ and thin‐layer chromatography on silica. The 4‐ and 5‐hydroxy acids forming the े‐ and ै‐lactones were separated from the other hydroxy acids by TLC on silica. The concentrations of a number of free and bound lactones in butterfat were determined by means of a radio‐gas chromatograph using the isotope dilution method. In addition the total concentration of the free and bound hydroxy acids which cannot be lactonized, was determined.