1. Single calcium-activated potassium channels (KCa channels) were recorded from membrane patches of rat olfactory bulb neurons in culture. Only one kind of KCa channel was seen, and it was present in approximately 50% of detached patches. 2. This channel, like maxi-KCa channels of other tissues, had a single-channel conductance of 270 pS, a reversal potential (Erev) of 0 mV in symmetrical K+, and was highly selective for K+ over Na+ and Cl-. 3. The KCa channel was blocked by d-tubocurarine (d-TC) on the cytoplasmic side, and charybdotoxin (CTX) on the extracellular side. This pharmacology is identical to that of one type of KCa channel from rat brain, observed previously in artificial bilayers and called the type 1 KCa channel. 4. The probability that the channel was in the open state (Po) increased with membrane depolarization. The position of the Po versus transmembrane voltage (Vm) curve was shifted by changes in [Ca2+]i so that the channel was open more often in higher [Ca2+]i. The gating kinetics resembled those of the type 1 KCa channel observed in bilayers. 5. Po was increased after superfusion of the cytoplasmic membrane surface with the active catalytic subunit of cyclic AMP-dependent protein kinase (PK-A), together with MgATP. Phosphorylation altered the distribution of channel closed times but had little effect on open times. The results suggest that phosphorylation is an important molecular mechanism in modulating the activity of this KCa channel from mammalian brain.