The biosynthetic pathway of chlorophylla-a formation in the photosynthetic phytoflagellate Englena gracilis was investigated by administration of 2H- and 13C-labeled precursors. L-[1-13C]Glutamate was incorporated into eight carbon atoms in the chlorophyll macrocycle via δ-aminolevulinic acid formed through the C-5 pathway. A low incorporation of D-[1-13C]glutamate was also observed. [2-13C]Glycine was preferentially incorporated into the methyl ester carbon of chlorophyll via the one-carbon metabolic pathway, but not via the Shemin pathway. Feeding experiments with 2H-, and 2H, 13C-labeled glycine yielded labeled chlorophylls, which were subjected to nuclear magnetic resonance and mass spectra analyses to obtain detailed information about the biosynthetic orgin of the hydrogen atoms.