1. Muscarinic K+ current (IK(ACh)) elicited by acetylcholine (ACh) was measured in guinea-pig atrial myocytes, which were either freshly isolated or cultured for up to 8 days. 2. The half-time of activation of inward IK(ACh) by a saturating concentration (10 microM) of ACh decreased from approximately 400 ms (in freshly isolated cells) to 250 ms after 6 days in culture. This was paralleled by an increase in the fast desensitizing component of IK(ACh). The density of steady-state currents was not changed. Downregulation of M2 receptors by long-term treatment of isolated myocytes with carbachol in vitro had opposite effects. 3. The EC50 of ACh for the activation of steady-state IK(ACh) was reduced from 5 x 10(-7) M (day 0) to 8 x 10(-8) M (day 6). The shift in EC50 occurred with a half-time of about 2 days, similar to the recovery from downregulation induced by treating atrial myocytes with carbachol in vitro. 4. The increase in sensitivity to ACh can be accounted for by an approximately 6-fold increase in the density of M2 receptors. 5. It is concluded that sensitization in culture reflects recovery from downregulation of M2 receptors due to the tonic vagal input to the heart in the intact animal.