Junctional and extrajunctional acetylcholine receptors were characterized in diaphragm muscle obtained from mature adult and aged rats. Rhodamine-conjugated alpha-bungarotoxin was used to visualize receptor localization. At this level of resolution, there were no major changes in receptor distribution, and nerve terminals were consistently associated with receptors and vice versa. Specific binding characteristics were assayed by measuring 125I-alpha-bungarotoxin binding. Maximal binding to intact junctional and extrajunctional tissue samples was greater in the older rats. The association rate constant in minced tissue decreased in the older animals. Retardation of the initial rate of toxin binding by d-tubocurarine was described by a two-component nonlinear Hofstee plot; values of Ki were about the same for both age groups, but there was a significant shift towards the low-affinity values in the aged rats. Miniature end-plate currents (m.e.p.c.s.) were recorded under voltage-clamp conditions before and after AChE inhibition. When AChE activity was inhibited m.e.p.c. amplitudes and decay time-constants increased in both age groups. The magnitude of these increases was larger in the older animals. Inhibition of AChE did not affect mean channel open time, which was estimated from spectral analyses of ACH-induced membrane noise. Lipid composition was assayed in whole muscle and isolated sarcolemma. Muscle cholesterol concentration rose 15-20 percent, but phospholipid concentrations were maintained. However, neither cholesterol, phospholipid levels, nor membrane fluidity changed significantly with age in isolated sarcolemmal membrane fractions. These data indicate that the numbers of junctional and extrajunctional receptors increase with age. In the junctional region, this is quite likely due to an expanded field of receptors and not an increased density. This is associated with an increased fraction of receptors with lower binding affinity during aging. These changes apparently are not caused by major changes in membrane fluidity or lipid composition.