Free 4-chlorotryptophan (4-Cl-Trp) was isolated from immature seeds of Pisum sativum, identified, and its stereochemistry determined to be L-form. Reinvestigation of the N-malonyl-4-Cl-Trp isolated from the same seeds showed that the stereochemistry of the 4-Cl-Trp residue is L-form. Chlorine-containing tryptophan (1) was first isolated from immature seeds of Pisum sativum as N-malonyl derivatives of 4-chlorotryptophan (2a, 2b), and its stereochemistry was assigned to be D-form¹). 4-Chloroindole-3-acetic acid (4-Cl-IAA) was also isolated from the same seeds as the second natural auxin (plant hormone)²), exhibiting much stronger activity—for example, more than one hundred times the inhibition of lettuce seed germination—than indole-3-acetic acid (IAA), the latter being the first natural auxin, now ubiquitously distributed in the plant kingdom. Although approximately sixty years have passed since the discovery of IAA in nature, its biosynthetic pathway is not fully elucidated, particularly whether it originates from L-tryptophan (L-Trp, 5a) or its D-isomer. Immature pea seeds, which produce both 4-Cl-Trp and 4-Cl-IAA along with minor amounts of Trp and IAA, could be a good biosystem to investigate the biosynthetic pathway of auxins (4-Cl-IAA and IAA) from their possible precursors (4-Cl-Trp or Trp). Recently, we found that 4-Cl-Trp isolated from the hydrolyzate of pea seed protein showed the L-form³). Therefore, N-malonyl-4-Cl-Trp should be reinvestigated for its absolute stereochemistry—whether it has the D-form as previously assigned⁴) or the L-form as present in the protein fraction. Here, we describe the isolation of free 4-Cl-Trp as well as N-malonyl-4-Cl-Trp from immature pea seeds and determine their absolute stereochemistry to both be L-form. These findings urge us to further reinvestigate the past assignment of the D-form to N-malonyl-Trp, which was reported in other plant species.