Abstract-The individual, deuterated, isomeric a-and p-carotenes were isolated from the green alga, Scenedesmus obliquus, cultivated in D,O containing 99.7 to 99.8 atom percent deuterium. Mass spectroscopy showed that both the a-and P-deuterio-carotene preparations contained principally the fully deuterated pigment molecules (C40D56), small quantities of deuterated molecules with one proton (C,,D,,H) and yet smaller quantities of deuterated molecules with two protons (C40D54H,). From statistical calculations the deuterio-carotene preparations also contained one to several isotopically-substituted deuterio-carotenes of each mass in the mass range 585 to 599 because of variation of the number of 13C and H atoms per molecule. The mass fragmentation of the deuterated pigments was analogous to that of the respective ordinary a-and @-carotene. It indicated that the protons in the C4,D,,H and C,,D,,H, molecules were distributed approximately randomly in various parts of the structure as in the terminal rings and in the ends and central portions of the polyene chain.WHEN autotrophic plants are grown in heavy water (99.7 to 99.8 percent D,O plus 0.3 to 0.2 percent H,O), the deuterated chloroplast pigments are f0rmed.l to The mass spectra of these pigments should establish the presence of the individual, fully-deuterated, molecular species. They should also reveal the presence and the proportions of any deuterated molecules containing protons. Moreover, they should indicate the number and the approximate location of these protons in the deuterated molecules. Comparison of these spectra with those of the ordinary hydrogen-containing pigments should reveal any effects of deuterium upon the fragmentation proces~es.~ to l1Of the three principal kinds of chloroplast pigments, the isomeric or-and P-carotenes were selected for comparative mass spectroscopic studies. These polyene hydrocarbons (C40H56) and their similar, deuterated analogs (C40D56) are the simplest chloroplast pigments. By contrast with the chlorophylls and xanthophylls these carotenes contain no protons or deuterons that are readily exchangeable with those of the solvents and sorbents employed for their extraction and isolation.There are many locations for the biosynthetic incorporation of protons into deuterated carotenes. As indicated by its structural formula, deuterio-j3-carotene, contains fourteen carbon atoms with a single deuteron each, six with two deuterons each and ten methyl groups with three deuterons each (see formula). Because of the symmetry of the molecule there are, however, only fourteen unique positions that may be occupied by a single proton. Some of these unique locations have many more deuterons than others. The geminate methyl groups, one pair in each ring, have twelve equivalent deuterons, but the 'in-chain' =CD-units have only two equivalent deuterons per pair. As shown by its unsymmetrical structural formula deuterio-or-carotene has twenty-nine discrete locations for the incorporation of a single proton. For dihydrogen substituents of deu...