In the present study we describe a high-performance liquid chromatographic method for the simultaneous determination of α-, β-, γ- and δ-tocopherol, cis and trans isomers of retinol, and total carotenes in different Italian cheeses. The method involves sample extraction and saponification followed by normal-phase high-performance liquid chromatography (HPLC). Monitoring of the absorbance and fluorescence is achieved using a programmable spectrophotometer and a programmable spectrofluorometer connected in series. The proposed method, which has the advantage of being able to evaluate simultaneously the fat-soluble vitamins considered by us in this work, has been applied to some Italian cheeses (Percorino romano, Taleggio, Provolone, and Caciotta) and milk samples (cows', goats', and sheep milk). The HPLC analytical procedure provides excellent reproducibility of results and is less subject to interferences from the sample matrix than wet chemical techniques. These factors combine to make the simultaneous determination of α-, β-, γ-, and δ-tocopherol, cis and trans isomers of retinol, and total carotenes by HPLC a practical technique for routine quality control and chemical and nutritional research applications.