Adrenal chromaffin cells synthesize and release catecholamines and several other transmitters that play important physiological roles in the coordinated response to stress or danger. The main trigger for secretion is acetylcholine (ACh) released from splanchnic nerve terminals that activates nicotinic ACh receptors (nAChRs) on the chromaffin cells, causing membrane depolarization and Ca2+ entry primarily through voltage-gated Ca2+ channels (Ca-channels). G protein-coupled receptors (GPCRs) can also trigger secretion, and it has been suggested that closure of M-type K+ channels might contribute to this process. However, GPCRs have multiple effects on calcium signaling and secretion, including release of intracellular Ca2+ stores, activation of second messenger pathways and kinases, and Ca2+ entry through store/receptor-operated channels. Hence, the effects of M-channel closure on [Ca2+]i signaling and transmitter release remain unclear. We have investigated the effects of linopirdine, a relatively selective blocker of M-channels, on stimulus-secretion coupling in chromaffin cells. Linopirdine produced a small increase in [Ca2+]i in approximately 63% of cells because of influx through Ca-channels. However, this was not sufficient to promote catecholamine release. We also show that linopirdine reduced cholinergic-stimulated increases in [Ca2+]i and secretion, primarily through potent block of nAChRs and a subtle effect on Ca2+ entry via Ca-channels. Hence, our data support the hypothesis that M-channels help control the excitability of chromaffin cells, but additional pathways need to be recruited by GPCRs to trigger catecholamine release. Furthermore, linopirdine potently targets nAChRs to modulate stimulus-secretion coupling in adrenal chromaffin cells.