A callus line of Vitex negundo was established using explants from the leaves. The calli showed stable morphology and thin layer chromatographic (TLC) profiles over several subcultures and in a variety of culture conditions. Seven oleanane-type triterpenes were identified from the callus culture extracts. The major triterpenes (2α,3α-dihydroxyolean-12-en-28-oic acid, 2α,3α,23-trihydroxyolean-12-en-28-oic acid, and oleanolic acid) were identified using 1 H and 13C NMR, MS and IR, while the minor triterpenes (2α,3α,23-trihydroxyolean-12-en-28-oic acid methyl ester, 11-oxo-olean-12-en-28-oic acid propyl ester, 11-oxo-olean-12-en-28-oic acid butyl ester, and β-amyrin) were identified through their EIMS fragmentations alone. A biosynthetic scheme for the formation of oleanane-type triterpenes in V. negundo is proposed.