Specific binding characteristics of acetylcholine receptors at the diaphragm neuromuscular junction of rats aged 10 (mature adult) and 28 (aged) months were assayed by measuring 125I-alpha-bungarotoxin binding. Maximal binding to intact tissue samples was greater in the older rats; this could be attributed to an age-related increase in terminal branching. The toxin concentration at which half-maximal binding occurred increased in the older rats. Binding kinetics were assayed in finely minced tissue samples, and the association rate constant was observed to decrease in the 28-month animals. Retardation of the initial rate of toxin binding by d-tubocurarine (dTC) in minced tissue was described by a two-component nonlinear Hofstee plot; IC50 values (7.1-7.2 microM and 39.0-46.5 nM) were about the same for both age groups, but there was a significant shift toward the low-affinity values in the aged rats. Rhodamine-conjugated alpha-bungarotoxin was used to visualize receptor localization. There were no major changes in receptor distribution, and nerve terminals were consistently associated with receptors and vice versa. The data indicate a shift toward lower binding affinity during aging, which may involve changes either in one of the two toxin-binding sites on individual receptors, in dTC blocking of the channel moiety, or in receptor types.