CALCIUM-DEPENDENT potassium (Kca) channels carry ionic currents that regulate important cellular functions1-3. Like some other ion channels4-10, Kca channels are modulated by protein phosphorylation2,11,15-21. The recent cloning of complementary DNAs encoding Slo Kca channels12-14 has enabled Kca channel modulation to be investigated. We report here that protein phosphorylation modulates the activity of Drosophila Slo KCa channels expressed in Xenopus oocytes. Application of ATP-γS to detached membrane patches increases Slo channel activity by shifting channel voltage sensitivity. This modulation is blocked by a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Mutation of a single serine residue in the channel protein also blocks modulation by ATP-γS, demonstrating that phosphorylation of the Slo channel protein itself modulates channel activity. The results also indicate that Kca channels in oocyte membrane patches can be modulated by an endogenous PKA-like protein kinase which remains functionally associated with the channels in the detached patch.