Activation of β-chemokine receptors, co-receptors for human immunodeficiency virus type-1 (HIV-1), stimulates movement and secretion in microglia, possibly through a Ca2+-dependent mechanism. We studied chemokine activation of Ca2+ signaling processes in microglia. Human fetal microglia were grown in primary culture and chemokine-induced increases in intracellular calcium concentration ([Ca2+](i)) were measured in single cells using indo-1-based microfluorimetry. Application of 50ng/ml regulated on activation, normal T expressed and secreted (RANTES; 120s) evoked responses in 26% of the microglia (187/719 cells). [Ca2+](i) increased from a basal level of 66±6nM to peak at 268±23nM (n=187). Chemokine-evoked responses rapidly desensitized as indicated by the rapid return to basal [Ca2+](i) levels in the maintained presence of RANTES. The removal of extracellular Ca2+ or stimulation in the presence of Ni2+ (2mM) or La3+ (100μM) blocked the RANTES-elicited [Ca2+](i) increase. The L-type calcium channel antagonist nimodipine (10μM) inhibited the RANTES-mediated increase in [Ca2+](i) by 80±16%. Thus, the RANTES-evoked calcium transient appears to result from Ca2+ influx with little if any release from intracellular stores. Application of gp120(clade) (E) and gp120(CM235) (50ng/ml) neither mimicked nor antagonized the RANTES-evoked response. Application of 50ng/ml eotaxin (120s) evoked an increase in [Ca2+](i) in 13% of the human microglia in culture (61/469 cells). The HIV-1 regulatory protein Tat (50ng/ml) increased the [Ca2+](i) in a subset of eotaxin-responsive cells (16/30). The L-type calcium channel antagonist nimodipine (3μM) inhibited eotaxin- and Tat-mediated increases in [Ca2+](i) by 88±6% and 93±6%, respectively. Thus, activation of CCR3 appears to evoke Ca2+ influx through L-type Ca2+ channels.These results indicate that β-chemokines, RANTES and eotaxin, activate a nimodipine sensitive Ca2+ influx pathway in human fetal microglia. HIV-1 Tat protein mimicked chemokine-mediated Ca2+ signaling and may modulate the migratory and secretory responses of microglia. Copyright (C) 2000 IBRO.