An efficient reversed-phase high-performance liquid chromatographic assay with ultraviolet detection at 450 nm for simultaneous determination of lutein and zeaxanthin stereoisomers in bovine retina is described. The procedure involves rapid and careful one-step hexane extraction of the carotenoids from the homogenized liquid and enrichment by on-line solid phase extraction on a polystyrene cartridge. The substances were eluted at a flow rate of 1 ml/min with acetone-water (85:15, v/v) on a C30 reversed-phase column. C30 phases exhibit superior shape selectivity for the separation of carotenoid stereoisomers compared with conventional C18 phases. For comparison a mixture of iodine-isomerized lutein and zeaxanthin standards was separated.