A reversed-phase high-performance liquid chromatography system for the complete separation of naturally occurring retinyl fatty acyl esters (RFAE) is described. The sensitivity of the method allows the detection of as little as 40 pmol of the various RFAE. The procedure was applied to the separation and estimation of endogenous RFAE present in tissues of normal rats; in addition, the incorporation of [3H]retinyl acetate into RFAE was also investigated. Retinyl palmitate is the major fatty acyl ester (79%) present endogenously in various tissues. However, eight other RFAE were also present in some tissues. At 24 hours after the injection of the label, radioactivity present in retinol and its metabolites was recovered mainly in liver tissue followed by kidneys, adrenals, lungs, intestine, trachea, testis, blood, heart and spleen. However, it was found that, in liver tissue, the specific radioactivity (dpm/nmol) of several RFAE was greater than that of retinyl palmitate (retinyl laurate 66-fold, retinyl pentadecanoate 5-fold, retinyl palmitoleate 4-fold).