Nesprin-1 and nesprin-2 (also known as Syne-1 and Syne-2,) are large ( approximately 3300-residue) vertebrate proteins associated with emerin and lamin A at the nuclear envelope of muscle cells and other cell types. We show that the previously described nesprins are short isoforms of giant proteins comprising an actin-binding amino-terminus connected to a carboxy-terminal klarsicht-related transmembrane domain by a massive ( approximately 6000-8000 amino acid) spectrin-like rod domain, making full-length nesprin-1, at one megadalton, the largest non-titin protein hitherto described in humans. We find that MSP-300, a 7000-residue Drosophila melanogaster protein whose disruption results in defects of muscle development, corresponds to the N-terminal two-thirds of the Drosophila nesprin ortholog. A nesprin-like protein is also encoded by the nematode genome. Moreover, we demonstrate that the larger isoforms of nesprin-1, like MSP-300, are localized to the sarcomeric Z-line of both skeletal and cardiac muscle. The recognition that a characteristic muscle-specific mutant phenotype in the fly results from a disruption of its nesprin ortholog reinforces the candidacy of the human proteins for involvement in genetic diseases of skeletal and cardiac muscle.