The amino groups of insulin have been studied by 'H and I3C nuclear magnetic resonance spectroscopy in insulin, zinc-free insulin and methylated insulin. By difference spectroscopy it is possible to follow the shift with pH of the r-CH2 and &CH2 proton resonances of lysine-B29 in insulin. In methylated insulin the dimethyl proton resonances of glycine-Al, phenylalanine-B1 and lysine-B29 can be followed as a function of pH. In native insulin pK,,, values of 6.7 and 8.0 are obtained for phenylalanine-B1 and glycine-A1 (the assignment is tentative) and 11.2 for lysine-B29. Separate resonances have been observed from the lysine-B29 NE-(CH3)2 group for the monomeric and dimeric forms of methylated insulin, which indicates a small change in the environment of lysine-B29 on dimerisation. The nuclear magnetic resonance spectral characteristics of these groups are, in general, consistent with the overall structure of the crystal form of the 2-zinc insulin hexamer .In recent years insulin has been subjected to intensive study from both the physiological and structurai standpomrs. Studies of the relationship between structure and function have been greatly aided and rationalized by the availability of a detailed crystal structure for the 2-zinc insulin hexamer [l]. On the other hand, a variety of studies summarized by Blundell et al. [l] suggests that the 2-zinc hexamer is not the physiologically active form, which is probably the monomeric or perhaps dimeric form of insulin. Furthermore, although many solution studies, in particular chemical modification results, can be rationalized from the crystal structure of the insulin 2-zinc hexamer, it is still not clear to what extent this structure is applicable to less aggregated forms of insulin in solution.With these points in mind we have undertaken a study of the amino groups of insulin by NMR spectroscopy. In particular, previously developed techniques for observation of methylated amino groups in proteins [2,3] have been applied to the study of the methylated amino groups of glycine-A 1, phenylalanine-B 1 and lysine-B29. In addition it is shown that application of 'H NMR difference spectroscopy [4] can be used to observe the methylene groups of lysine in unmodified proteins (see also [5]). The ability to observe the various insulin amino groups has led to a detailed analysis of the pH-dependent behaviour of the amino groups of glycine-Al, phenylalanine-B1 Abbreviation. NMR, nuclear magnetic resonance and lysine-B29. This has not been possible in previous 19F and I3C NMR studies of insulin derivatives, in which the amino groups have been trifluoroacetylated [6,7] or carbamylated with '3C-enriched potassium cyanate [8]. In the discussion we present an analysis of the relationship between the pH-dependent behaviour of these amino groups and the crystal structure of the 2-zinc insulin hexamer. This analysis suggests that while the overall insulin conformation for less aggregated forms in solution is probably similar to that found in the crystal structure, a conformational ch...