It is believed that the nicotine concentration in tobacco is closely correlated with the amount of nitrogen (N) supplied. On the other hand, N uptake mainly occurs at the early growth stage, whereas nicotine concentration increases at the late growth stage, especially after removing the shoot apex. To identify the causes of the increased nicotine concentration in tobacco plants, and to compare the effects of different ways of mechanical wounding on nicotine concentration, field experiments were carried out in Fuzhou, Fujian Province in 2003 and 2004. Excision of the shoot apex had almost no influence on N content in the plant; however, it caused dramatic increases in nicotine concentration in leaves, especially in the middle and upper leaves. An additional increase of the nicotine concentration was obtained by removal of axillary buds. The wounding caused by routine leaf harvests, however, did not change the leaf nicotine concentration, and neither did reducing leaf harvest times. The present results revealed no direct relationship between N supply and nicotine concentration in tobacco leaves, and indicate that not all kinds of mechanical wounding were capable of stimulating nicotine synthesis in tobacco plants. Since nicotine production is highly dependent on the removal of apical meristems and hence on the major sources of auxin in the plant, and application of 1-naphthylacetic acid onto the cut surface of the stem after removing the shoot apex markedly decreased the nicotine concentration in different leaves and the total nicotine content in the plant, the results suggest that decreased auxin supply caused by removal of the shoot apex as a kind of mechanical wounding might regulate nicotine synthesis in the roots of tobacco plants.