31P NMR studies have been performed on vegetative and encysted Acanthamoeba castellanii. A prominent feature of the spectra is the presence of resonances characteristic of P-C linkages. Vegetative cells show sharp phosphonate resonances whereas encysted cells show broad phosphonate resonances and narrow phosphate resonances. Studies of model compounds using 31P NMR have shown the narrow resonances to arise from 2-aminoethylphosphonic acid; the broad components from encysted cells arise from molecules with long rotational correlation times such as aggregates of lipophosphonoglycan or phosphonoprotein. Vegetative cells also show the presence of an electronegatively beta-substituted phosphonic acid, possibly 1-hydroxy-2-aminoethylphosphonic acid, a component identified in the cell membrane lipophosphoglycan. pH titration studies of a number of phosphonic acid model compounds show a chemical shift behaviour quite distinct from that of biological phosphates. Upon acidification from pH 12, resonances shift to high field followed by return to lower field at pH 1.0. These resonances can be ambiguous as monitors of cellular pH. The pH of both vegetative and encysted cells is 6.0--6.5. Encysted cells under anaerobic conditions show no spectral changes over 24 h. Vegetative cells under similar conditions show time-dependent changes characteristic of variation in cellular pH. There appears to be little correlation between pH and the variously differentiated states (vegetative or encysted) in Acanthamoeba.