The plant tryptophan (Trp) biosynthetic pathway produces many secondary metabolites with diverse functions. Indole-3-acetic acid (IAA), proposed as a derivative from Trp or its precursors, plays an essential role in plant growth and development. Although the Trp-dependant and Trp-independent IAA biosynthetic pathways have been proposed, the enzymes, reactions and regulatory mechanisms are largely unknown. In Arabidopsis, indole-3-glycerol phosphate (IGP) is suggested to serve as a branchpoint component in the Trp-independent IAA biosynthesis. To address whether other enzymes in addition to Trp synthase alpha (TSA1) catalyze IGP cleavage, we identified and characterized an indole synthase (INS) gene, a homolog of TSA1 in Arabidopsis. INS exhibits different subcellular localization from TSA1 owing to the lack of chloroplast transit peptide (cTP). In silico data show that the expression levels of INS and TSA1 in all examined organs are quite different. Histochemical staining of INS promoter-GUS transgenic lines indicates that INS is expressed in vascular tissue of cotyledons, hypocotyls, roots and rosette leaves as well as in flowers and siliques. INS is capable of complementing the Trp auxotrophy of Escherichia coliDeltatrpA strain, which is defective in Trp synthesis due to the deletion of TSA. This implies that INS catalyzes the conversion of IGP to indole and may be involved in the biosynthesis of Trp-independent IAA or other secondary metabolites in Arabidopsis.