Objective - To evaluate whether biochemical or genetic alterations in AMP-activated protein kinase (AMPK) play a role in the development of polysaccharide storage myopathy (PSSM) in Quarter Horses. Animals - 30 PSSM-affected and 30 unaffected (control) Quarter Horses. Procedures - By use of an established peptide phosphotransfer assay, basal and maximal AMPK activities were measured in muscle biopsy samples obtained from 6 PSSM-affected and 6 control horses. In 24 PSSM-affected and 24 control horses, microsatellite markers identified from the chromosomal locations of all 7 AMPK subunit genes were genotyped with a fluorescent DNA fragment analyzer. Alleles of 2 of the AMPK γ subunit genes were genotyped via DNA sequencing. Allele frequencies of DNA markers in or near the AMPK subunit genes were measured in isolated genomic DNA. Results - No differences in basal or maximal muscle AMPK enzyme activities between PSSM-affected and control horses were detected. There were also no differences in allele frequencies for microsatellite markers near any of the 7 AMPK subunit genes between the 2 groups. Furthermore, previously known and newly identified alleles of 2 equine AMPK γ, subunit genes were also not associated with PSSM. Conclusions and clinical relevance - These results have provided no evidence to indicate that AMPK plays a causative role in PSSM in American Quarter Horses.