Thymopoietin or TMPO (indicated by its alternative gene symbol, LAP2, in this work) has been proposed as a candidate disease gene for dilated cardiomyopathy (DCM), since a LAP2 product associates with nucleoplasmic lamins A/C, which are encoded by the DCM gene LMNA. We developed a study to screen for genetic mutations in LAP2 in a large collection of DCM patients and families. A total of 113 subjects from 88 families (56 with familial DCM (FDC) and 32 with sporadic DCM) were screened for LAP2 mutations using denaturing high‐performance liquid chromatography and sequence analysis. We found a single putative mutation affecting the LAP2α isoform in one FDC pedigree. The mutation predicts an Arg690Cys substitution (c.2068C>T; p.R690C) located in the C‐terminal domain of the LAP2α protein, a region that is known to interact with lamin A/C. RT‐PCR, Western blot analyses, and immunolocalization revealed low‐level LAP2α expression in adult cardiac muscle, and localization to a subset of nuclei. Mutated Arg690Cys LAP2α expressed in HeLa cells localized to the nucleoplasm like wild‐type LAP2α, with no effect on peripheral and nucleoplasmic lamin A distribution. However, the in vitro interaction of mutated LAP2α with the pre‐lamin A C‐terminus was significantly compromised compared to the wild‐type protein. LAP2 mutations may represent a rare cause of DCM. The Arg690Cys mutation altered the observed LAP2α interaction with A‐type lamins. Our finding implicates a novel nuclear lamina‐associated protein in the pathogenesis of genetic forms of dilated cardiomyopathy. Hum Mutat 26(6), 566–574, 2005. © 2005 Wiley‐Liss, Inc.