The product of the X-linked Emery-Dreifuss muscular dystrophy gene is a single-membrane-spanning protein called emerin, which is localized to the inner nuclear membrane of all tissues studied. To examine whether a number of the mutant forms of emerin expressed in patients are mislocalized, we transfected GFP-emerin cDNA constructs reflecting these mutations into undifferentiated C2C12 myoblasts and showed that both wild type and all the mutant emerins are targeted to the nuclear membrane, but the mutants to a lesser extent. Mutant Del236-241 (deletion in transmembrane region) was mainly expressed as cytoplasmic aggregates, with only trace amounts at the nuclear envelope. Complete removal of the transmembrane region and C-terminal tail relocated emerin to the nucleoplasm. Mutations in emerin's N-terminal domain had a less severe effect on disrupting nuclear envelope targeting. This data suggests that emerin contains multiple non-overlapping nuclear-membrane-targeting determinants. Analysis of material immunoisolated using emerin antibodies, from either undifferentiated C2C12 myoblasts or purified hepatocyte nuclei, demonstrated that both A- and B-type lamins and nuclear actin interact with emerin. This is the first report of proteins interacting with emerin. The EDMD phenotype can thus arise by either the absence or a reduction in emerin at the nuclear envelope, and both of these disrupt its interactions with that of structural components of the nucleus. We propose that an emerin-nuclear protein complex exists at the nuclear envelope and that one of its primary roles is to stabilize the nuclear membrane against the mechanical stresses that are generated in muscle cells during contraction.