Compound 48/80 and 14C-labeled compound 48/80 were synthesized, and fractionated by thin-layer chromatography into 14 components (A-N) with various histamine releasing activities and different Ca2+ requirements for their actions. The histamine release induced from rat mast cells in vitro by the most active component, fraction D (molecular weight = 2280, a tridecamer composed of 13 monomer units), was greatly enhanced by extracellular Ca2+, and was partially reduced by pretreatment of the cells with dinitrophenylated ascaris antiserum, an IgE. In contrast, the histamine release induced by fraction H (molecular weight = 1580, a nonamer composed of 9 monomer units), was higher in Ca2+ -free medium than in Ca2+-containing medium, and was partially reduced by pretreatment of mast cells with neurotensin or substance P, Ca2+-independent releasers. Apparently both fractions D and H are useful reagents for investigating the role of Ca2+ in histamine release and releaser binding in mast cells.