The tryptic fragments of natural human adrenocorticotrophin, were separated by countercurrent distribution and a correction in positions 25, 26, 27 and 30 was made by Riniker et al. (1972) in a study of the fragment containing residues 22-39. We have purified the remaining tryptic fragments, namely residues 1-8, 9-15, 16-21 and 17-21, by using ion-exchange chromatography on CM-cellulose and have carried out sequence determination by using the subtractive Edman degradation procedure and digestion with aminopeptidase M and carboxypeptidase B. These results have confirmed the proposed sequence for human adrenocorticotrophin in regions 6-7, 10-14 and 17-20, which had previously been arrived at only by analogy with the invariant sequence found in the three other mammalian adrenocorticotrophin species that had been investigated.