Heritable deficiency of phosphorylase kinase (Phk), a regulatory enzyme of glycogen metabolism, is responsible for 25% of all cases of glycogen storage disease and occurs with a frequency of -1 in 100,000 births. It is genetically and clinically heterogeneous, occurring in X-linked and autosomal-recessive forms and exhibiting various patterns of principally affected tissues (liver only, muscle only, liver and muscle, liver and kidney, heart only). This heterogeneity is thought to reflect the enzyme's structural complexity [subunit composition, (alpha beta gamma delta)4] and isoform diversity. Two isoforms encoded by separate genes are known for the subunits alpha (muscle [alpha M] and liver [alpha L isoforms) and gamma (muscle [gamma M] and testis [gamma T] isoforms), whereas only one gene appears to exist for the subunit beta. The subunit delta is calmodulin; identical calmodulins are expressed from three different human genes. Additional isoform diversity arises by differential mRNA splicing of the alpha M, alpha L and beta subunits. Mutations responsible for the various forms of Phk deficiency are sought in those subunit/isoform genes with a matching chromosomal location and tissue-specificity of expression. We report here that autosomal liver-specific Phk deficiency is associated with mutations in the gene encoding the testis/liver isoform of the catalytic gamma subunit (PHKG2). We found homozygous PHKG2 mutations in three human patients of consanguineous parentage and in the gsd (glycogen storage disease) rat strain, which is thus identified as an animal model for the human disorder. One human mutation is a single base-pair insertion in codon 89 that causes a frameshift and premature chain termination. The three other mutations result in non-conservative replacements of amino acid residues (V106E, G189E, D215N) that are highly conserved within the catalytic core regions of all protein kinases. These are the first mutations to be reported for an autosomal form of Phk deficiency. The findings suggest that the PHKG2 gene product is the predominant isoform of the catalytic gamma subunit of Phk not only in testis but also in liver, erythrocytes and, possibly, other non-muscle tissues.