Abstract: A reverse- phase high performance liquid chromatographic method wasestablished for the simultaneous determination of trimethoprim (TMP), sulfadiazine (SD)and sulfamethoxazole (SMZ) in dissolution media. Meanwhile, another reverse- phasehigh performance liquid chromatographic method was developed for the simultaneous deter-mination of the three drugs mentioned above and their metabolites, N⁴- acetyl - SD andN⁴- acetyl-SMZ in serum. The in vitro dissolution and in vivo bioavailability of twocommercial trimethoprim- sulfadiazine- sulfamethoxazole tablets (A and B) werestudied. Drugs were released far more rapidly from A than from B. Significant difference(P<0.001)was observed between the T₅₀ s of the same active principles released from Aand B. The in vivo processes of all three active principles in 8 healthy subjects taking twotablets each could be described by one- compartment model with first - order absorptionand elimination. Compared with A, the relative bioavailabilities of B were 0.83,0.73and 0.78 for TMP, SD and SMZ, respectively. Drugs were absorbed more rapidly fromA than from B.