The negative chronotropic effect of acetylcholine (ACh) on the sinoatrial node fades in the continuous presence of ACh as a result of desensitization. We have investigated the mechanism underlying desensitization in single rabbit sinoatrial node cells using the whole cell patch clamp technique. The negative chronotropic effect resulting from the injection of a constant hyperpolarizing current faded. ACh activated an inwardly rectifying potassium current (iK,ACh), which faded in the continuous presence of ACh. ACh had no effect on "basal" L-type calcium current (iCa), but ACh decreased iCa, which had been potentiated by isoprenaline. This effect did not fade during a 2-min exposure to ACh. ACh decreased the hyperpolarization-activated current (i(f)). This effect again did not fade. These results suggest that desensitization of the negative chronotropic response to ACh is, in part, the result of the membrane hyperpolarization and, in part, the result of the fade of iK,ACh. These results also suggest that, whereas the activation of potassium current by ACh rapidly fades, the effects resulting from the inhibition of adenylate cyclase do not.