The effects of 2-alkanols as mobile phase modifiers on the resolution of 11-cis-, 13-cis-, 9-cis- and all-trans-retinol in high-performance liquid chromatography were studied. Partisil-10-ODS and Zorbax CN columns were used in series. The retentions of 9-cis-and all-trans-retinol were significantly influenced by the presence of long-chain 2-alkanols in the mobile phase. Baseline separation of 9-cis-and all-trans-retinol was achieved when 1% 2-heptanol in hexane was used as the eluent. On the other hand, the separation of 11-cis-from 13-cis-retinol required 5% dioxane in hexane. The method is applicable to the separation of retinol isomers present in biological samples. Analysis of normal rat liver lipids showed the presence of a small amount of 13-cis-retinol in addition to all-trans-retinol.