By incorporating plasma membrane vesicles into planar lipid bilayers, we previously characterized a family of four types of Ca(2+)-activated K+ channels from rat brain (Reinhart et al., 1989). Two of these are “large-conductance” or “maxi”-K+ channels, which differ in their gating kinetics and toxin sensitivity and are henceforth referred to as “type 1” and “type 2” channels. Here we show that the gating of these two channel types can be modulated by phosphorylation and dephosphorylation. The effects of cAMP-dependent protein kinase catalytic subunit (PK-A) on type 1 maxi-K+ channels are complex in that, while half of these channels are upregulated by the kinase, about one out of seven channels is downregulated. Thus, there may be several distinct channels within the type 1 category. Type 2 maxi-K+ channels are consistently downregulated by PK-A. The effects of PK-A on both channel types are reversed by the catalytic subunit of protein phosphatase 2A (PP-2A), but not by protein phosphatase 1 (PP-1). Furthermore, some of the type 1 maxi-K+ channels can be modulated by PP- 2A, even without any prior PK-A treatment, indicating they are in a phosphorylated state when they are incorporated into the bilayer. The results demonstrate that (1) type 1 and type 2 maxi-K+ channels are substrates for PK-A; (2) phosphorylation can shift the open probability of channels in either direction, by a mechanism involving multiple phosphorylation sites; (3) phosphorylation alters the Ca2+/voltage sensitivity of these channels; and (4) dephosphorylation of type 1 and type 2 channels is catalyzed by specific phosphatases.