Activation of brain macrophages (microglia) occurs rapidly following stimulation with lysophosphatidylcholine (LPC). We identified the physiological mechanisms underlying LPC-induced de-ramification and release of interleukin-1 in the murine microglia cell line BV-2. Patch-clamp experiments revealed activation of non-selective cation currents and Ca²⁺-dependent K⁺ currents by extracellular LPC. LPC-activated non-selective cation channels were permeable for monovalent and divalent cations. They were inhibited by 100 μM Gd³⁺ (n=11), 100 μM La³⁺ (n=10), 500 μM Zn²⁺ (n=4) and Grammostola spatulata venom (diluted 1:2000; n=7), but were unaffected by 100 μM diltiazem (n=7), 50 μM LOE908MS (n=7), 1 mM amiloride (n=7) and 200 μM DIDS (n=7). Ca²⁺ influx through non-selective cation channels caused sustained increases in intracellular Ca²⁺ concentration (n=168). These Ca²⁺ increases were sufficient to elicit charybdotoxin-sensitive Ca²⁺-dependent K⁺ currents (n=21). In LPC-stimulated microglial cells, non-selective cation currents caused transient membrane depolarization, which was followed by sustained membrane hyperpolarization induced by Ca²⁺-dependent K⁺ currents (n=9). Furthermore, 15 μM LPC elicited K⁺ efflux by stimulating electroneutral K⁺-Cl^– co-transporters, which were inhibited by 1 mM furosemide and 100 μM DIOA (n=4 experiments). LPC-induced morphological changes (de-ramification) were Ca²⁺-independent (n=150). They were prevented by simultaneous inhibition of non-selective cation channels and K⁺-Cl^– co-transporters (n=314; p<0.001). In contrast, Ca²⁺ influx through non-selective cation channels was required for LPC-induced release of interleukin-1 from microglial cells (n=4 experiments; p<0.001). Microglial IL-1 release was inhibited by 200 nM charybdotoxin (n=4 experiments; p<0.05), but was unaffected by 1 mM furosemide (n=4 experiments). These data suggest that distinct physiological mechanisms are involved in LPC-induced microglial activation.