Effects of guanyl nucleotides on the affinity of cholinergic agonists at the [3H]quinuclidinylbenzilate binding site have been compared with their effects on the intact left atrium. The binding of carbachol was described by a two-site model; guanyl nucleotides had little effect on the affinity at either site but increased substantially the proportion of low affinity sites. The negative inotropic effect of acetylcholine (ACh) was unaffected by exogenous guanyl nucleotides or variation in external Mg++ or Ca++. ACh (2 microM) shortened the action potential, reduced spike amplitude and usually increased the maximum diastolic potential. GTP and guanyl-5'-yl-imidodiphosphate in the presence of ACh increased action potential amplitude and duration and increased further the diastolic potential. Low external Mg++ had similar effects to guanyl-5'-yl-imidodiphosphate but their effects were not additive. It is suggested that these compounds delay the activation of a transient outward K+ current by ACh.