The activated proteinase inhibitor α₂-macroglobulin (α₂M*) binds to two receptors, the low density lipoprotein receptor-related protein (LRP-1) and the α₂M* signalling receptor (α₂MSR). Silencing LRP-1 gene expression in macrophages by RNA interference does not block α₂M* activation of signalling cascades. We now demonstrate that transfection of macrophages with a double-stranded RNA homologous in sequence to the Grp78 gene markedly decreased induction of inositol 1,4,5-trisphosphate (IP₃) and subsequent IP₃-dependent elevation of [Ca²⁺]_i induced by α₂M*. Concomitantly, α₂M*-induced increase in [³H]thymidine uptake was abolished in these transfected cells. Insulin treatment significantly upregulates α₂MSR and it also caused a marked increase in Grp78 expression which could be blocked by RNA interference. α₂M* treatment of cells activates the Ras- and PI 3-kinase-dependent signalling pathways. Suppressing Grp78 expression leads to the loss of these activation events in transfected macrophages. We thus conclude that Grp78 is the α₂M* signalling receptor.