Microglial cells are activated in response to brain insults; the mechanisms of this process are not yet understood. One of the important signaling mechanisms that might be involved in microglia activation is related to changes in the intracellular calcium concentration ([Ca2+]i). Using fluo-3 microfluorimetry, we have found that external application of the complement fragment C5a (4-10 nM) induced [Ca2+]i elevation in microglial cells in situ in corpus callosum slices. Similarly, application of complement fragments C5a (0.1-10.0 nM) or C3a (100 nM) generates biphasic [Ca2+]i transients composed of an initial peak followed by a plateau in cultured microglia. Incubation of microglial cells for 30 min with pertussis toxin (PTX; 1 microgram/ml) inhibited both C5a- and C3a-triggered [Ca2+]i responses, suggesting the involvement of PTX-sensitive G-proteins in the signal transduction chain. Removal of Ca2+ ions from the extracellular solution eliminated the plateau phase and limited the response to the initial peak. The restoration of the extracellular Ca2+ concentration within 30-60 sec after the beginning of the complement fragment-induced [Ca2+]i elevation led to the recovery of the plateau phase. Inhibition of the endoplasmic reticulum Ca2+ pumps with 500 nM thapsigargin transiently increased the [Ca2+]i and blocked the [Ca2+]i signals in response to subsequent complement fragment application. Our data suggest that complement factors induce [Ca2+]i responses by Ca2+ release from internal pools and subsequent activation of Ca2+ entry controlled by the filling state of the intracellular Ca2+ depots.