Mammalian glycogen phosphorylases comprise a family of three isozymes, muscle, liver, and brain, which are expressed selectively and to varying extents in a wide variety of cell types. To better understand the regulation of phosphorylase gene expression, we isolated partial cDNAs for all three isozymes from the rat and used these to map the corresponding genes in the mouse. Chromosome mapping was accomplished by comparing the segregation of phosphorylase restriction fragment length polymorphisms (RFLPs) with 16 reference loci in a multipoint interspecies backcross between Mus musculus domesticus and Mus spretus. The genes encoding muscle, liver, and brain phosphorylases (Pygm, Pygl, and Pygb) are assigned to mouse chromosomes 19, 12, and 2, respectively. Their location on separate chromosomes indicates that distinct cis-acting elements govern the differential expression of phosphorylase isozymes in various tissues. Our findings significantly extend the genetic maps of mouse chromosomes 2, 12, and 19 and can be used to define the location of phosphorylase genes in man more precisely. Finally, this analysis suggests that the previously mapped "muscle-deficient" mutation in mouse, mdf, is closely linked to the muscle phosphorylase gene. However, muscle phosphorylase gene structure and expression appear to be unaltered in mdf/mdf mice, indicating that this mutation is not an animal model for the human genetic disorder McArdle's disease.