The electrochemical behaviour of vitamin D3and 25-hydroxyvitamin D3(25-OH D3) in a high performance liquid chromatography system using amperometric detection is described. Separation is carried out using a C18 reversed-phase column and the optimum mobile phase was a 0.1 M LiClO4solution in methanol-water (97:3, v/v) at a flow rate of 1.25 ml/min. 25-OH D3and vitamin D3were eluted with good resolution at retention times of 3 and 6 minutes respectively, and determined by amperometric detection with a glassy carbon electrode at + 1.050 V (vs Ag/AgCl). Calibration graphs for both substances showed good linearity when amounts of vitamin D3between 18 and 312 ng and 27 and 412 ng of 25-OH D3were injected. Detection limits of 8 ng (vitamin D3) and 25 ng (25-OH D3); relative standard deviations of 3.2% (vitamin D3) and 5.8% (25-OH D3) were obtained.