A simple, robust and reproducible analytical method for the determination of phosphocreatine (PCr), creatine (Cr) and creatinine (Cn) in equine skeletal muscle is presented. The technique used isocratic reverse-phase ion-pairing high-performance liquid chromatography. Neutralized perchloric acid extracts of equine muscle biopsies were analysed and the values obtained were compared with determinations from an established enzymic procedure. Good resolution of all three metabolites was achieved within a retention time of less than 11 min. Linearity for each metabolite within the concentration range in the samples was demonstrated. Peak purity was specifically addressed. The abolition of each creatine in a pooled extract by enzymic incubation showed no underlying peaks. It was concluded that peaks were free of co-eluents which would otherwise lead to an overestimation of PCr, Cr and Cn concentrations.