The effects of sodium nitroprusside (SNP) on Ca²⁺-dependent K⁺ (KCa) channels in cultured bovine adrenal chromaffin cells were investigated using single channel recording patch-clamp techniques. KCa channels were activated by application of 100 μM SNP to the extracellular side of cell-attached patches. Methylene blue (300 μM), an inhibitor of soluble guanylate cyclase, or H-8 (1 μM), a protein kinase inhibitor with relative specificity for cGMP-dependent protein kinase, diminished but did not completely abolish the SNP-induced KCa channel activation. Diethylamine/NO complex (DEA/NO), an NO donor, also activated KCa channels in cell-attached patches. Furthermore, application of 100 μM SNP or 100 nM DEA/NO to the intracellular surface of excised inside-out patches also activated KCa channels in the bath solution which contained 1 mM Ca²⁺. These results indicate that SNP is capable of activating the KCa channel via cGMP-dependent and -independent mechanisms. These studies demonstrate that NO may serve as an important regulatory mechanism for catecholamine secretion in chromaffin cells via the activation of KCa channels.