In search of vascular smooth muscle cell differentiation markers, we identified two genes encoding members of a new family of type II integral membrane proteins. Both are ubiquitously expressed, and tissue-specific alternative mRNA initiation and splicing generate at least two major isoforms of each protein, with the smaller isoforms being truncated at the N-terminus. We have named these proteins nesprin-1 and -2 for nuclear envelope spectrin repeat, as they are characterized by the presence of multiple, clustered spectrin repeats, bipartite nuclear localization sequences and a conserved C-terminal, single transmembrane domain. Transient transfection of EGFP-fusion expression constructs demonstrated their localization to the nuclear membrane with a novel C-terminal, TM-domain-containing sequence essential for perinuclear localization. Using antibodies to nesprin-1, we documented its colocalization with LAP1, emerin and lamins at the nuclear envelope, and immunogold labeling confirmed its presence at the nuclear envelope and in the nucleus where it colocalized with heterochromatin. Nesprin-1 is developmentally regulated in both smooth and skeletal muscle and is re-localized from the nuclear envelope to the nucleus and cytoplasm during C2C12 myoblast differentiation. These data and structural analogies with other proteins suggest that nesprins may function as ‘dystrophins of the nucleus’ to maintain nuclear organization and structural integrity.