Simple and ion-pair reverse phase high performance chromatographic separations combined with selective extraction were developed in order to achieve a qualitative and quantitative analysis of [3H]chloramphenicol (CP) metabolites in rat urine. Complete separation followed by unequivocal identification of all the metabolites was obtained, especially of the toxicologically significant CP-arylamine. Metabolic profiles were established from human and goat urine which confirmed data already available, but was used to identify new acidic metabolites in the latter species. This methodology appears to be a useful research tool in comparative metabolic studies.