Although it has been shown that glycine is the nitrogenous precursor of the protoporphyrin moiety of hemoglobin (1,2), it has not been previously established that glycine is utilized for the formation of all four pyrrole structures of the porphyrin. In protoporphyrin (Fig. 1) two of the pyrrole rings (I and II) contain methyl and vinyl side chains and two (III and IV) contain methyl and propionic acid side chains; it is conceivable that these two different pyrrole structures are synthesized in the animal organism from different precursors. Degradation experiments were therefore undertaken to study the distribution of isotopic nitrogen in N15-labeled heme, produced in vivo after the administration of N15-labeled glycine, in order to determine whether glycine is used in the synthesis of both pyrrole types. The degradation studies were carried out in a manner to give unequivocal data with respect to separation of the two different pyrrole structures found in protoporphyrin. Labeled hemin, obtained from the blood of a human and from the blood of ducks, after the administration of N16-labeled glycine, was converted into hematoporphyrin dimethyl ether which was subsequently oxidized to yield methylmethoxyethylmaleimide (from pyrrole rings I and II) and methyl propionic acid maleimide or hematinic acid (from pyrrole rings III and IV) (Fig. 1). It can be seen from Table I that in each experiment the N15 values of the methylmethoxyethylmaleimide from pyrrole rings I and II and of the hematinic acid from pyrrole rings III and IV were equal, and were identical with the Nls concentrations in the porphyrin. Therefore, these experiments demonstrate that glycine is utilized equally for the formation of the two types of pyrroles in the protoporphyrin. These findings are in harmony with the hypothesis that in the biosynthesis of protoporphyrin a pyrrole ring is formed which is a common precursor of both types of pyrrole structures found in the protoporphyrin. These studies do not as yet reveal the mechanism by which glycine is utilized in the biosynthesis of the porphyrin. However, this report, together with our previous work, demonstrated directly that the nitrogen of