We quantitated the concentrations of the principal organophosphate metabolites present in the intact crystalline rabbit lens, measured the intralens pH, and evaluated dynamic changes during 24 hr incubations, using phosphorus-31 nuclear magnetic resonance (P-31 NMR) spectroscopy. Tissue perchloric acid extracts prepared from these same lenses were analyzed by this technique to verify metabolite identifications and to quantitate the concentrations of the minor lens metabolites. Values for lens organophosphate concentrations, including three groups of previously unidentified phosphorus-containing substances, were established for freshly excised lenses, 24 hr incubated lenses, and lenses incubated in glucose-deficient media. Lens metabolite levels were not adversely affected by incubation in a medium previously shown to maintain lens clarity and ion transport capabilities. Conversely, lens incubation in glucose-deficient media induced significant metabolic changes characterized by a time-dependent decline in ATP, corresponding increases in ADP, inorganic phosphate, and phosphorylated hexoses. Cataract formation was noted after incubation in this medium. These findings support the hypothesis that alterations in the organophosphate levels of the lens actually preceded changes in the Na+ and K+ concentrations and therefore may be the "initiating factor" in formation of lens cataracts.