The amino acid sequence of the 146 residues of the beta-chain of the major haemoglobin from the platypus has been determined. The soluble peptides derived from the chain by tryptic digestion were isolated by paper ionophoresis and chromatography. The amino acid sequences were determined by the dansyl-Edman procedure or by further digestion with other enzymes. The tryptic peptides were aligned by homology with other beta-globins. There were 14 changes in sequence compared with echidna beta-chain. The number of changes in sequence compared with human beta-chain is 34 which is less than the 39 changes between human and platypus alpha-chains. Generally there are more changes between beta-chains; there are only three other examples reported where there are more changes between alpha-chains than beta-chains, these are of echidna, rabbit and dog globins. By comparison with the 'contact sites' in horse haemoglobin there is one change in beta-haem contacts, three changes in beta1-alpha1 contacts and no changes in beta2-alpha1 contacts. The date of divergence of the monotremes from the other mammals was estimated at 132 +/- 33 million years, based on the number of amino acid differences between species and allowing for multiple mutations during the evolutionary period. This estimate differs widely from the estimate given by similar treatment of the alpha-chain sequences and the significance of this discrepancy to the validity of the method is discussed.