Multinucleated giant cells, characteristic of granulomatous infections, originate from fusion of macrophages, however, little is known about the underlying mechanism. Alternative activation of macrophages by exposure to IL-4 and IL-13 induces macrophage homokaryon formation. We have established a new quantitative bifluorescent system to study IL-4-induced fusion of primary murine macrophages in vitro. Using this assay, we could show that macrophage fusion is not mediated by a single molecule, but involves multiple functional components. Although several murine macrophage populations were not competent to form giant cells, indicating that they fail to display the full fusion machinery, these non-fusogenic macrophages could fuse with fusion-competent macrophages in a heterophilic manner. Since IL-4 induced molecules were needed on both fusion partners, we conclude that at least two functionally distinct molecules mediate macrophage homokaryon formation with each present on one fusion partner. In addition, though IL-4 treatment led to induction of a fusogenic status, macrophages could only fuse efficiently when adherent to a permissive substratum. Based on our findings, we conclude that macrophage fusion is a multistage process involving multiple target molecules. The model we describe will allow analysis of the molecular basis of membrane fusion and possible insight into alternative activation of macrophages.