A high-performance liquid chromatographic procedure was developed for purification and quantification of alpha-tomatine from tomatoes. A crude preparation of glycoalkaloids from tomato fruits was fractionated on a column of Nucleosil NH2 (10 mu m), and eluted with a mixture of tetrahydrofuran/acetonitrile/0.02 M KH2PO4 (50/30/20, v/v/v). The column effluent was monitored at 205 nm. Total analysis of alpha-tomatine was completed in < 10 min. It was present at highest concentrations in fruit at the mature-green stage but its level decreased rapidly as fruit approached full maturity. The technique should be useful for analysis of alpha-tomatine in biological samples.