The role of methyltransferase (MT) reactions in acetylcholine (ACh)-evoked depolarization and contraction in primary chick myotubes was determined by using a mixture of erythro-9-(2-hydroxy-3-nonyl) adenine, homocysteine thiolactone, and adenosine which together inhibit the activity of MT. Carboxy-MT and lipid-MT activities were inhibited irreversibly by 91 and 100%, respectively. ACh-induced muscle contraction was also inhibited within 10 min after application of the inhibitor mixture. However, in contrast to permanent inhibition of MT activities, inhibition of acetylcholine receptor (AChR)-dependent muscle contraction was reversible. Moreover, physiological studies showed that the inhibitor mixture had no effect on resting membrane potential or ACh-induced depolarization or desensitization. These results suggest that AChR function is not altered by methylation inhibitors and that changes in AChR-mediated contraction are not due to inhibition of MT activities.