Torpedo shaped embryos dissected from young fruits of Boerhaavia diffusa were cultured in semisolid MS basal medium supplemented with 2,4-D and BAP either alone or in various combinations. Callus from the embryos was transferred to fresh medium of the same composition after 20 days. The callus when initiated was creamish, compact to friable and fast growing. With passage of time the callus growth became slow and the callus became brown. Cytological studies revealed that at the end of 30 days in culture most of the callus was composed of elongated cells which were either nucleated or enucleated. Light microscopic studies indicated depositions on the surface of these cells. The brown callus was dried and extracted in 50 % ethanol. The extract was re-extracted with pure ethanol. UV scanning of the ethanolic extract gave a twin absorption peak typical of flavonoids. HPTLC of the extract performed against kaempferol as standard revealed the presence of kaempferol at 1.53 μg/mg of callus. The study demonstrated that callus cells growing in vitro are able to differentiate and activate the flavonoid biosynthetic pathway resulting in the production of the important flavonoid, kaempferol.