Three new copies of a sesquiterpenoid synthase gene encoding 5-epi-aristolochene synthase (EAS) were cloned as cDNAs from Nicotiana attenuata and functionally characterized after expression of the recombinant enzymes in E. coli. Differential patterns of EAS gene expression and formation of the antimicrobial sesquiterpenoid capsidiol were found in roots and shoots of two species of Nicotiana. EAS is expressed constitutively in roots of N. attenuata and N. sylvestris corresponding with constitutive capsidiol formation in roots. Constitutive expression of EAS and capsidiol accumulation were not detectable in shoots of rosette plants of N. attenuata, but accumulation of terpene synthase transcripts could be induced in shoots by feeding of the tobacco hornworm, Manduca sexta. Constitutive expression of EAS has not been previously reported from N. tabacum, where capsidiol is known as an elicitor- and pathogen-inducible phytoalexin. It is conceivable that capsidiol contributes not only to an inducible defense against pathogens, but also to a constitutive defense in an organ-specific manner in some species of Nicotiana.