The ability of cloned human alpha2B-adrenoceptors heterologously expressed in Sf9 cells and endogenous alpha2B-adrenoceptors in NG 108-15 neuroblastoma x glioma cells to couple to increase of intracellular Ca2+ was studied. Ca2+ increases in NG 108-15 cells were detectable but slight, whereas those in alpha2B-adrenoceptor-expressing Sf9 cells were greater. In the latter, the maximum Ca2+ increase correlated positively, and the EC50-value of noradrenaline negatively, with the receptor expression density. The order of potency of the agonists was D-medetomidine ([D]-4--[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole) > noradrenaline approximately = clonidine > oxymetazoline, with clonidine and UK14,304 (5-bromo-N-[4,5-dihydro-1H-imidazole-2-yl]-6-quinoxalinamine) being weak partial agonists. In Sf9 cells Ca2+ increases consisted of concomitant mobilization from an intracellular store and influx of extracellular Ca2+. In these cells alpha2B-adrenoceptor stimulation also increased the inositol 1,4,5-trisphosphate mass. We conclude that alpha2B-adrenoceptors can couple to intracellular Ca2+ increases which may involve prior activation of phospholipase C.