Summary: Ruta graveolens utilizes anthranilate synthase (AS) for the synthesis both of tryptophan in primary metabolism and acridone alkaloids in secondary metabolism. AS has been purified from plants and cell cultures of R. graveolens 670‐ and 1700‐fold, respectively. Glutamine‐ and ammonia‐ dependent AS activities were strictly co‐purified in all steps. Through cDNA cloning and complementation of Escherichia coli deletion mutants defective for AS, it is shown that young Ruta plants express two genes for functional ASα subunits, ASα1 and ASα2. The data indicate that ASα from Ruta requires an ASβ subunit with a native molecular weight of 60–65 kDa for the glutamine‐dependent reaction. Protein synthesized invitro from cloned cDNA is processed upon import into isolated chloroplasts, indicating that mature ASα subunits are active in plastids in vivo. ASα1 and ASα2 are constitutively expressed in Ruta cell cultures, but ASα1 steady‐state mRNA levels are increased 100‐fold 6 h subsequent to elicitation whereas ASα2 expression remains constitutive. Increased ASα1 transcription corresponds to elicitor‐induced alkaloid accumulation. The data indicate that Ruta regulates anthranilate flux into primary and secondary metabolism through differential regulation of AS genes specific to these pathways.