Background: VSM excitability is regulated by large conductance, calcium-activated K+ (BK) channels. NO elevates VSM cytosolic cGMP levels, and activates the type I cGMP-dependent protein kinase (cGKI) to phosphorylate the BK channel at key residues. The nature and location of residues phosphorylated remain unresolved, along with their functional importance to the channel enhancement process. Purpose: To identify key phosphorylatable residues underlying the NO/cGMP/cGKI-mediated BK channel enhancement in VSM and correlate their functional contribution with their phosphorylation status. Procedures: Proximity ligation assay (PLA) was used to show BKα subunit and cGKI protein co-localization (spatial restriction <40 nm). In vitro phosphorylation of immune-purified wild type (WT, GenBank Acc# U09383) BK channel by cGKIα was characterized biochemically using radiolabeled γ32ATP to identify phosphorylated residues using a combination of phospho-amino acid (AA) analysis and peptide mapping. Identified residues were individually replaced and mutant channels were transiently-transfected into A7r5 cells (Lipofectamine method), and studied under cell-attached patch clamp using 1s voltage ramps (-30mV to 200mV, 0mV holding potential). Results: Freshly isolated rat cerebral VSM cells exhibited PLA fluorescence indicating BKα-cGKI association. Extensive association was also observed in A7r5 cells transiently-expressing BK channels. Non-transfected cells showed no measurable fluorescence. Biochemical characterization of BK channels phosphorylated by purified cGKI in vitro revealed 32P-labeled phosphoserine residues at “Arg/Lys-Arg/Lys/X-X-pSer” locations (where X is any AA). Using these criteria, 9 potential Ser residues were identified within the BKα subunit (see Figure 1). For each location, the critical Ser residue was mutated to Ala and mutant channels were expressed in A7r5 cells for functional analysis. Phosphorylation of WT BK channels by cGKI using γ32P-ATP revealed a major ~120 kDa 32P-labelled band corresponding to the BKα subunit. However, the 32P-labeling intensity was clearly reduced in 3 mutant channels (25%, 50% and 60% reductions at Ser691, Ser873 and Sers1111-1113, respectively), indicating multiple phosphorylation sites. Recordings from A7r5 cells expressing WT BK channels confirmed current enhancement to NO/cGMP/cGKI pathway stimulation (spermine-nonoate, 5 μM; db-cGMP, 100 μM). However, BK channels containing Ser to Ala substitutions at each of the 3 sites exhibiting low 32P-labeling intensities exhibited no enhancement. This study uncovers a definitive molecular mechanism explaining the BK channel enhancement following cGMP production in situ.