Plasma disposition, protein binding, urinary recovery, and renal clearance of sulfamethazine (SMZ), sulfamerazine (SMR), and sulfadiazine (SDZ) and their N4-acetyl and hydroxy derivatives were studied in 4 horses in a crossover trial. The plasma concentration-time curves of the metabolites paralleled those of the parent drug in the elimination phase. Sulfamethazine and SMR were extensively metabolized. In plasma and urine, the main metabolite of the 3 sulfonamides tested was the 5-hydroxypyrimidine derivative, which was highly glucuronidated. Difference in elimination half-life of SMZ, SMR, and SDZ could be related to difference in metabolism and renal clearance values. Metabolism speeds drug elimination, producing compounds with higher renal clearance values than those of the parent drug. Methyl substitution in the pyrimidine side chain increased hydroxylation of the parent drug, but prolonged the persistence of the sulfonamides studied in the body. The high concentration of N4-acetyl and hydroxy metabolites of SMZ and SMR in plasma and urine decreased the potential antibacterial activity of the parent drugs. Sulfadiazine was less metabolized, and microbiologically determined SDZ concentrations in plasma and urine were slightly lower than those measured by high-performance liquid chromatography.