1. Depolarization of glial cells causes their intracellular pH (pHi) to increase. To more completely characterize this depolarization-induced alkalinization (DIA) in mammalian astrocytes, we studied DIA in cultured rat hippocampal astrocytes. Astrocytes were loaded with the fluorescent pH indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), and pHi was monitored with the use of an imaging system. Cells were studied approximately 24 h after removing them from serum-containing culture medium. In HCO-3-buffered solution containing 3 mM K+, mean baseline pHi was 7.14 +/- 0.14 (mean +/- SD). 2. Astrocyte pHi rapidly and reversibly alkalinized when bath [K+] was increased from 3 to 12 mM. In HCO-3-buffered solution, mean DIA amplitude was 0.16 +/- 0.01 pH units, and mean rate of pHi change was 0.076 +/- 0.03 pH units/min. In contrast, DIA elicited in nominally HCO-3-free, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution was much smaller (0.03 +/- 0.01 pH units, 0.04 +/- 0.01 pH units/min; P < 0.0001), indicating that DIA was, in large part, a HCO-3-dependent process. Subsequent experiments were carried out in HCO-3-buffered solution. 3. The relationship between DIA and variable changes in bath [K+] was examined. Increasing bath [K+] from 3 to 6 mM produced a DIA of 0.07 +/- 0.04 pH units, and lowering [K+] to 0.5 mM resulted in an acid shift of 0.08 +/- 0.05 pH units. The effects of these changes in [K+] on membrane potential were measured in separate experiments by whole cell patch-clamp recording. On the basis of these data, it was possible to construct a relationship between Vm and pHi; shifting membrane potential approximately 10 mV resulted in a pHi shift of approximately 0.07. 4. Application of 0.5 mM Ba2+ depolarized Vm and elicited DIA in astrocytes. This indicated that depolarization, in the absence of an increase in [K+], could cause DIA. Application of Ba2+ also blocked K(+)-induced DIA, presumably because blockade of K+ channels prevented any depolarization by K+. 5. Cells with more alkaline baseline pHis exhibited larger and more rapidly developing DIAs. The mechanism of this effect is not known. 6. The timing of serum removal affected astrocyte DIA. Cells studied approximately 24 h after serum removal always exhibited robust DIA (mean = 0.16 +/- 0.01 pH units).(ABSTRACT TRUNCATED AT 400 WORDS)