An HPLC method for MAs (mugineic acid and its analogues) analysis was established. By this method, time course change of MAs secreted from the roots of Fe‐deficient barley were traced. After about one week of ‐Fe treatment, MAs secreted from the roots gradually increased and after 40 days it became plateau. The several MAs were synchronously secreted from the roots throughout a day; their amounts were maximum at noon. The pattern of MAs secretion was different from that of amino acids. MAI was found to be trace in roots, xylem sap and shoots of the control (+Fe) plant. However, MAs concentrations remarkably increased in those tissues when Fe‐deficiency chlorosis progressed due to ‐Fe treatment. From experiments of 14CO2‐fixation by photosynthesis, 14C‐MA1 secreted from the roots was found to be biosynthesized with a half‐life of 24 h.