The 31 P nuclear magnetic resonance spectrum of liquid milk was examined. Of the three peaks observed, the two larger were assigned to inorganic phosphate (Pj) and the seryl phosphate (SerP) residues of casein; the third peak was assigned to a phosphodiester, which is probably glycerophosphoryl choline. The pH-dependences of the chemical shifts of the Pj and SerP were measured with and without added EDTA, and the results confirm the assignments. The width of the P t peak in milk is significantly greater than in similar solutions lacking casein, probably because of binding to, or chemical exchange with, the casein micelle. Most of the SerP residues in milk are not sufficiently mobile to have been detected in these experiments but a significant fraction of SerP residues is able to move freely and can be titrated.In recent years there has been growing interest in the applications of nuclear magnetic resonance (NMR) spectroscopy to biological systems (Gadian, 1982). Of particular interest has been the use of 31 P high resolution NMR with samples in aqueous solution and also, more recently, with solid state samples (Herzfeld et al. 1980;Tropp et al. 1983). For experiments in the solution state, the nuclei of interest are excited with a pulse of radio-frequency energy, after which the response of the system is recorded. Fourier transformation of the data creates the spectrum in the frequency domain. For biological systems, it is the non-invasive nature of such experiments, with little or no sample preparation, that makes NMR so attractive a technique. The conditions of the experiment are such that only the ions or molecules that contain mobile 31 P atoms are observed, while, in general, immobile species give rise to spectra with lines too broad to be observed. Thus, when applied to biological systems that are metabolizing, various phosphorus-containing compounds, such as adenosine triphosphate, phosphocreatine, inorganic phosphate (Pj) (Gadian et al. 1979), can be recognized. On the other hand, structures such as bone or phospholipid membranes, containing relatively immobile phosphorus atoms, are not observed; however, phosphoproteins in certain conditions may give distinguishable resonances, as for example in the case of phosvitin (Colman & Gadian, 1976;Belton et al. 1983) and casein (Ho et al. 1969;Humphrey & Jolley, 1982). A particularly useful feature of the 31 P resonance from both Pj and phosphoseryl residues in phosphoproteins is that their chemical shifts are pH-dependent and can give direct information on their state of ionization. This has proved a very valuable method for measuring %