The two major urinary metabolites of 14C-timolol in man, involving oxidation and hydrolytic cleavage of the morpholine ring, are also observed in both Sprague-Dawley rats and CRCD-1 mice. These are the N-T(4-[3-(1,1-dimethylethyl)amino]-2-hydroxypropoxy)-1,2,5-thiadiazol-3-ylT-N-(2-hydroxyethyl)glycine and 1-(1,1-dimethylethylamino-3-([4-(2-hydroxyethylamino)-1,2,5-thiadiazol-3-yl]oxy)-2-propanol. The former was previously identified erroneously as the isomeric compound 1-(1,1-dimethylethylamino)-3-([4-(N-2-hydroxyethylglycolamido)-1,2,5-thiadiazol-3-yl]oxy)-2-propanol. Rats and mice had two additional metabolites in common, 1-[(1,1-dimethylethyl)amino]-3-([4-(2-hydroxy-4-morpholinyl)-1,2,5-thiadiazol-3-yl]oxy)-2-propanol and a compound now proposed to be the corresponding morpholino lactone 1-[1,1-dimethylethyl)-amino]-3-([4-(2-oxo-4-morpholinyl)-1,2,5-thiadiazol-3-yl]oxy)-2-propanol but for which the corresponding isomeric morpholino lactam structure 1-[(1,1-dimethylenthyl)-amino]-3-([4-(3-oxo-4-morpholinyl)-1,2,5-thiadiazol-3-yl]oxy))-2-propanol was tentatively proposed in an earlier publication. A metabolite observed in the rat, but not in the other species studied, was 4-(4-morpholinyl)-1,2,5-thiadiazol-3-ol-1-oxide. The metabolic pattern in these rodents does not change significantly after repeated doses. A scheme summarizing the metabolic fate of timolol in man and laboratory animals is presented.