Extracts of medicinal plants were examined for the effects on the NGF-mediated neurite outgrowth from PC12D cells to evaluate their NGF-potentiating activities. In the methanol extracts, Gymmopteris rufa (LINN.) BERNH, Ruta graveolens LINN. and Picrorhiza scrophulariiflora PENNELL increased markedly the proportion of the neurite-bearing cells. In the case of ethyl acetate fractions. Equisetum giganteum LINN. produced the most powerful enhancement of the proportion of the neurite-bearing cells. In the water fractions. Imperata cylindrica, Ginseng Radix, Gymmopteris rufa (LINN.) BERNH, Gochnatia polymorpha (LESS) CAB and Picrorhiza scrophulariiflora PENNELL caused a weakly enhancement of the proportion of PC12D cells with neurites. We successfully isolated nardosinone, picrosides I and II. gersemiol and 9-hydroxysemperoside aglucone as enhancers of NGF-action. We studied the effects of nardosinone. picrosides. gersemiol and 9-hydroxysemperoside aglucone on NGF-induced neurite outgrowth and associated transmembrane signal transduction in PC12D cells. These compounds did not exhibit the neurotrophic activity but caused a concentration-dependent enhancement of the NGF-induced neurite outgrowth from PC12D cells. Nardosinone- and picrosides-induced enhancements of the NGF-action were abolished by GF109203X, a protein kinase C inhibitor. Furthermore, PD98059. a potent MAPK kinase inhibitor, completely blocked nardosinone- and picrosides-induced enhancements of the neurite outgrowth in the presence of NGF. These results suggest that nardosinone and picrosides activate PKC-MAPK-dependent signaling pathway. Interestingly, no increases in expression of phosphorylated MAPK were observed in nardosinone and picrosides-treated PC12D cells in the presence of NGF. On the basis of pharmacological data, it is suggested that nardosinone, picroside I or II enhances the NGF-induced neurite outgrowth from PC12D cells probably by amplifying a down-stream step of MAPK in the NGF receptor-mediated intracellular PKC-MAPK-dependent signaling pathway.