An automated pre-column high-performance liquid chromatographic method has been developed for the isolation of adibendan and metabolites from biological fluids and for their simultaneous quantitative assay. High sensitivities were obtained by the use of a multiple-injection device allowing solid-phase extraction from several successive sample injections with enrichment of metabolite traces on the pre-column. Two metabolites in dog urine were identified as N-oxypyridine (M1) and 2-hydroxypyridine (M2) derivatives of adibendan, while the structure of M3 is still unknown. M1 and M2 are also metabolites in rats, rabbits and humans, and contribute to cardiovascular efficacy. The metabolic profiles were determined in plasma, urine and bile, as a function of dose, route of administration and sex, using radioactivity and ultraviolet detection of the eluates.