A new method for the determination of naturally occurring imino acids in plants was devised. This method comprises the formation of dinitrophenyl derivatives and their measurement by HPLC. The determination limits were 100 pmol for 4-hydroxypipecolic acid (HPA) and proline (Pro) and 10 pmol for pipecolic acid (Pip). The recoveries of HPA, Pro, and Pip from leaf homogenate of an Acacia species were 95.9 ± 2.8, 100.5 ± 1.7, and 101.1 ± 2.3%, respectively. HPA was found in leaves, seeds, and roots of five kinds of Acacia but not in leaves of other members of the legume family. The HPA content of Acacia dealbata link leaves was much higher in winter than in summer. From X-ray analysis, HPA was found to be hydrated by two water molecules that are situated in a vacant channel formed by the molecular arrangement of HPA. HPA had no remarkable physiological or pharmaceutical activities.