The regulation of calcium-activated potassium (KCa) channels by endogenous protein kinase A (PKA) was examined in inside-out patches from equine tracheal myocytes. In the absence of exogenous protein kinases, ATP (500 microM) significantly augmented KCa channel activity when applied to the cytosolic patch surface [open-state probability (nP0, mean +/- SE) increased from 0.010 +/- 0.001 to 0.034 +/- 0.005 (n = 24)]. The stimulatory effect of ATP was mimicked by ATP-gamma-S but not by AMP-PNP. Rather, AMP-PNP significantly inhibited channel activity. The PKA inhibitory peptide (PKI) significantly reduced nPo and prevented the augmenting effect of subsequent ATP. Ht 31, an inhibitory peptide for A-kinase-anchoring proteins (AKAP), but not its proline-substituted mutant, also blocked the stimulatory effect of ATP. These results suggest that 1) ATP augments KCa channel activity through phosphorylation; 2) the phosphorylation is catalyzed by endogenous PKA; 3) anchoring via AKAP is required to maintain association of PKA with the membrane; and 4) in a newly obtained patch, some of the KCa channels are probably already in a phosphorylated state.