Cobalt-57-bleomycin is a diagnostically useful radiopharmaceutical, but little is known about the nature of its individual fractions in regard to their metal-binding capacity and their in vivo distribution. Bleomycin was separated by high performance liquid chromatography (HPLC) into four major components. These were labeled and the distribution studied in tumor-bearing rats at 2 and 24 hr. In vivo radiochemical purity was also determined. Of the nine HPLC systems studied, Porasil A eluted with a mobile phase of 0.3% ammonium formate in methanol gave the best separation of the fractions. These fractions were copper free and retained their biologic activity and purity. An in vitro competitive binding study of 57Co-bleomycin with either 57Co-human serum albumin (HSA) or 57Co-ethylenediaminetetraacetic acid (EDTA) showed the labeled bleomycin to be a strong chelate. The biologic distribution in tumor-bearing rats showed significantly higher concentration in tumors at 2 hr for fractions A2 and B2 as compared to the bleomycin mixture. The other fractions, A1 and demethylA2, gave lower tumor concentrations than the bleomycin mixture. The tumor-to-blood ratios for A2 and B2 were not significantly different from the bleomycin mixture, suggesting that the concentration of the bleomycin in the tumor was related to blood concentration. Tumor-to-blood ratios of greater than 10:1 at 2 hr were achieved for A2, B2, and the mixture; ratios of greater than 31:1 were achieved at 24 hr for all three. From these data it appears that the major components A2 and B2 are the most useful for diagnostic tumor imaging.