The effect of several second messengers on the functional expression of gap junctions was investigated in primary cultures of newborn rat microglia. As previously reported, microglia cultured under resting conditions expressed low levels of the gap junction protein connexin 43, and exhibited little dye coupling. After treatment with 4bromo-A23187, a Ca(2+) ionophore, the incidence of dye coupling between microglia increased progressively over a 12-h period. Dye coupling was markedly reduced by gap junction blockers. Induction of dye coupling by 4bromo-A23187 was prevented by the addition of a synthetic peptide with the same sequence as a region of the extracellular loop 1 of connexin 43 (residues 53-66). The increase in dye coupling induced by 4bromo-A23187 was associated with increased connexin 43 mRNA and protein levels. Treatment of microglia with phorbol 12-myristate 13-acetate, an activator of protein kinase C, did not promote gap junctional communication in untreated microglia and reversed 4bromo-A23187-induced dye coupling. Thus, gap junctional communication between microglia can be regulated oppositely by calcium- and protein kinase C-dependent pathways. Activators of cGMP-dependent protein kinase (8bromo-cGMP) or protein kinase A (8bromo-cAMP) had no effect on untreated microglia or on 4bromo-A23187-induced dye coupling. Differential regulation of gap junctions by intracellular calcium concentration and protein kinase C activity may help to explain how various stimuli evoke differences in microglia responses, such as synthesis and secretion of cytokines and proteases.