Secretory vesicles isolated from rat liver were found to fuse after exposure to Ca2+. Vesicle fusion is characterized by the occurrence of twinned vesicles with a continuous cleavage plane between two vesicles in freeze-fracture electron microscopy. The number of fused vesicles increases with increasing Ca2+-concentrations and is half maximal around 10(-6)M. Other divalent cations (Ba2+, Sr2+, and Mg2+) were ineffective. Mg2+ inhibits Ca2+-induced fusion. Therefore, the fusion of secretory vesicles in vitro is Ca2+ specific and exhibits properties similar to the exocytotic process of various secretory cells. Various substances affecting secretion in vivo (microtubular inhibitors, local anesthetics, ionophores) were tested for their effect on membrane fusion in our system. The fusion of isolated secretory vesicles from liver was found to differ from that of pure phospholipid membranes in its temperature dependence, in its much lower requirement for Ca2+, and in its Ca2+-specificity. Chemical and enzymatic modifcations of the vesicle membrane indicate that glycoproteins may account for these differences.