Neomycin, an antibiotic previously thought to interact specifically with inositol-containing phospholipids, was found to inhibit IP 3 -mediated Ca 2 + release from the intracellular stores of permeabilized insulinoma and liver cells. This inhibition could be relieved by increasing the IP 3 concentration. Radiolabelled IP 3 was found to bind tightly to columns prepared from neomycin covalently attached to glass beads. ATP was also bound by these colums. It is concluded that neomycin acts in biological systems as a weak anion exchanger and is therefore unsuitable for use as a specific tool to study the role of inositol phospholipids in intracellular signalling.