We have used whole-cell and single-channel recording techniques to investigate the electrophysiological properties of cultured ovine oligodendrocytes (OLGs). Our studies have led to the following conclusions. (1) Cultured mature OLGs express a variety of voltage-dependent K+ conductances including an outward current that consists of a transient component and a steady-state component, as well as an inwardly rectifying K+ current. (2) These conductances are expressed sequentially as a function of development in culture. The inwardly rectifying K+ current appears later than the outward current. (3) Although process extension may influence the expression of the ion channels, the majority of the K+ channels are located in the soma of OLGs, probably concentrated in the basal plasma membrane. (4) Finally, the activation of K+ channels in OLGs can be inhibited by two distinct second messengers, cAMP acting through protein kinase A and diacylglycerol acting through protein kinase C, the effects of which perhaps converge at the level of a common phosphorylated enzyme or regulatory protein. Both cAMP and diacylglycerol have been implicated as factors important in controlling the induction of a myelinogenic metabolism associated with OLG substratum attachment. Thus, membrane ion channels may provide an important intermediate step linking cellular substratum attachment to the eventual induction of myelinogenesis.