A method for the quantitative determination of vitamin B6 in food by using high-pressure liquid chromatography and a fluorescence detector is described. The analytical method measures any vitamin B6 compound present in food (pyridoxamine, pyridoxal, pyridoxine and their phosphoric acid esters). Pyridoxamine (PM), pyridoxal (PL) and pyridoxine (PN) are released from the food after 30 min hydrolysis with 0.2 N-sulfuric acid at 120 degrees C. The filtered extract is applied directly to a reversed phase Spherisorb ODS column. PM, PL and PN are separated as mobile phase by using 0.08 N-sulfuric acid and measured by means of the fluorescence detector (excitation: 290 nm, emission: 395 nm). Recovery rates of PM, PL and PN added to the samples were between 94 and 102% depending on the kind of food. The detection limit is 5 micrograms PM and 10 micrograms PL or PN, resp. per 100 g food. The analytical procedure is simple, specific and has many applications; the results are well reproducible.