Myophosphorylase deficiency in cattle is a muscle disease induced by a C-->T point mutation in codon 489 of the myophosphorylase gene, which until now has only been diagnosed in the Charolais breed. The disease seems to be inherited in an autosomal monogenic recessive manner. A calf of double muscled phenotype was suspected of suffering from myophosphorylase deficiency based on typical symptoms, i.e. brown-coloured, transparent urine, occurring after exercise; exercise intolerance; symptoms of pain; and an elevated level of plasma creatine kinase. The presence of the previously described mutation was excluded using a newly developed, improved polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) procedure to identify easily heterozygous carriers and homozygous affected animals.