Agrin, an extracellular matrix protein involved in neuromuscular junction formation, directs clustering of postsynaptic molecules, including acetylcholine receptors (AChRs). This activity resides entirely in the C-terminal portion of the protein, which consists of three laminin-like globular domains (G-domains: G1, G2 and G3) and four EGF-like repeats. Additionally, alternate mRNA splicing yields G-domain variants G2(0,4) with 0- or 4-amino-acid inserts, and G3(0, 8,11,19) with 0-, 8-, 11- or 19-amino-acid inserts. In order to better understand the contributions of individual domains and alternate splicing to agrin activity, single G-domains and covalently linked pairs of G-domains were expressed as soluble proteins and their AChR clustering activity measured on cultured C2 myotubes. These analyses reveal the following: (1) While only G3(8) exhibits detectable activity by itself, all G-domains studied (G1, G2(0), G2(4), G3(0) and G3(8)) enhance G3(8) activity when physically linked to G3(8). This effect is most pronounced when G2(4) is linked to G3(8) and is independent of the order of the G-domains. (2) The deletion of EGF- like repeats enhances activity. (3) Increasing the physical separation between linked G1 and G3(8) domains produces a significant increase in activity; similar alterations to linked G2 and G3(8) domains are without effect. (4) Clusters induced by two concatenated G3(8) domains are significantly smaller than all other agrin forms studied. These data suggest that agrin G-domains are the functional units which interact independently of their specific organization to yield AChR clustering. G-domain synergism resulting in biological output could be due to physical interactions between G-domains or, alternatively, independent interactions of G-domains with cell surface receptors which require spatially localized coactivation for optimal signal transduction.