The actin-myosin contractile apparatus consists of several thick filament and thin filament proteins. Specific regulatory mechanisms are involved in this highly ordered process. In this paper, we reported the identification and characterization of a novel myofibrillogenesis regulator, MR-1. The MR-1 gene was cloned from human skeletal muscle cDNA library by using a strategy that involves EST data base searching, PCR and RACE. The MR-1 gene is located on human chromosome 2q35 and encodes a 142 aa protein. Northern blot revealed that the mRNA level of MR-1 was highest in the skeletal muscle and certain level of MR-1 expression was also observed in heart, liver and kidney. Immunohistochemical assay confirmed that the MR-1 protein existed in human myocardial myofibrils. It was found by yeast two-hybrid screening and confirmed by in vitro binding assay that MR-1 could interact with sarcomeric proteins, such as myosin regulatory light chain, myomesin 1 and beta-enolase. These studies suggested that MR-1 might play a regulatory role in the muscle cell and it was worth investigating further.