Rippling muscle disease (RMD) is an autosomal dominant disorder characterized by electrically silent, percussion-induced muscular contractions. We previously reported the localization of a gene for RMD to 1q41-q42 by genome-wide linkage analysis in a large family from Oregon. This RMD gene was initially found to be contained within a 12-cM interval with a maximum multipoint lod score of 3.56. A YAC/BAC contig was assembled by STS content mapping and database searches spanning the nonrecombinant interval containing the RMD gene (RMD1). The physical map, in conjunction with recent mapping information from various other sources, clarified the order of genetic markers in this region and necessitated redefinition of the RMD genetic interval by linkage analysis with the newly ordered markers. Polymorphisms that mapped to the YACs in this contig were genotyped in this family and used to provide statistical support for narrowing of the critical genetic interval to 3 cM, corresponding to a maximum possible physical distance of 4.0 Mb. In addition, recombination breakpoint mapping supported the evidence that RMD1 must reside within this interval between markers D1S446 and D1S2680. ESTs (82) were mapped to the YACs spanning the region known to contain the RMD1 gene, and of these, 9 become strong positional candidates. The physical and refined genetic maps of this RMD locus set the stage for isolation of the responsible gene and elucidation of a novel patho-mechanism of calcium homeostasis in skeletal muscle.