1. The balance between acetylcholine (ACh) synthesis and degradation and the availability of choline were studied in the neuromuscular junction of rats aged 10 (mature adult) and 28 (aged) months. Endogenous and 2H4-labelled variants of ACh and choline were assayed during steady-state stimulated and resting conditions using gas chromatography-mass spectrometry. 2. Endogenous ACh levels were 34% less per nerve terminal in the older rats; in non-innervated tissue, the levels were 12-14% as large as in innervated tissue. Endogenous choline levels in innervated and non-innervated tissue were similar and were 28% higher in the older animals. 3. Accumulation of 2H4-labelled choline was significantly greater in the 28-month animals; the specific activity at equilibrium was 60% higher in the older rats. 4. Incorporation of [2H4]choline into [2H4]ACh was significantly faster in the aged rats; times to half-maximal values were 2.0 and 0.6 min in the 10- and the 28-month animals, respectively. There was no measurable synthesis of ACh in the non-innervated tissue. 5. There were no major age-related differences in the amount of ACh released during stimulation between 1 and 20 Hz. In contrast, choline efflux during stimulation was significantly greater in the aged animals; flux values (+/- S.E. of mean) were 0.48 (+/- 0.014) and 0.66 (+/- 0.038) nmol/min for the 10- and the 28-month rats, respectively. 6. Under resting conditions, endogenous ACh efflux was significantly greater in the older rats; the rates (+/- S.E. of mean) were 2.00 (+/- 0.21) and 3.05 (+/- 0.43) pmol/min for the younger and the older animals, respectively; similarly, choline efflux was greater in the aged tissue. 7. These results indicated that lower intracellular ACh levels can be attributed to enhanced leakage rather than to decreased synthesis. Greater ACh efflux is accompanied by correspondingly greater choline uptake in the 28-month rats; this is associated with greater choline efflux and higher steady-state choline levels in the aged tissue.