The results of previous structural studies of bleomycin are summarized in Fig. 1. In this communication, we propose the total structure of bleomycin as shown in Fig. 2. Potentiometric titration of copper-free bleomycin B2 hydrochloride revealed three basic functions with pK'a values of 7.3, 4.7, and approximately 2.9, along with a terminal guanidino group with a pK'a > 11.5. Treatment of bleomycin with 2,4-dinitrofluorobenzene followed by acid hydrolysis indicated that the α-amino group of the β-aminoalanine moiety is free, while its secondary amine forms an amide. Proton NMR and UV spectroscopy assigned the pK'a 4.7 function to the imidazole ring and the pK'a ~2.9 function to the 4-aminopyrimidine moiety. Elemental analysis supported the molecular formula of copper-free bleomycin B2 hydrochloride (C55H81N19O21S2·3HCl), and 13C-PFT-NMR spectroscopy confirmed the carbon number (C55). Methylation experiments and the synthesis of β-aminoalanine-α-betaine amide verified that the carboxyl group of the β-aminoalanine moiety exists as an amide in the bleomycin molecule. The IR spectrum of bleomycin was consistent with the proposed structure, particularly the β-lactam ring, which exhibited a bathochromic shift due to intramolecular hydrogen bonding. Furthermore, the structural relationship between bleomycin and phleomycin was established via the oxidative transformation of phleomycin D1 to bleomycin B2, allowing us to propose the structure of phleomycin as shown in Fig. 5.