In this paper a HPLC method for the determination of lutein, zeaxanthin, beta-cryptoxanthin, alpha-carotene, beta-carotene and lycopene in mixed vegetables and fruit and in human plasma is described. The carotenoids were well separated and the separation was achieved within fifteen minutes using a HPLC system consisting of a 5 microns Vydac 201TP54C18 column, an UV detector, methanol-tetrahydrofuran (95:5 v/v) as mobile phase and a flow rate of 1.0 ml/min. The validity of the separation method was determined by evaluating the linearity of the calibration graphs of each carotenoid (between 0.1 and 1.0 microgram/ml for all compounds except lycopene between 0.1 and 0.8 microgram/ml, r = 0.999) and the accuracy of the chromatographic response (CV < 10%). The reproducibility of the retention times was also good. In the foods samples the extraction procedure was very effective whereas, the saponification step significantly damaged some of the carotenoids. In the plasma the extraction and separation of these compounds were also effective and the qualitative data obtained comparable with those reported in literature. The use of echinenone as internal standard helped to improve quality control.