The mechanism of inhibition of human thyroid iodide peroxidase (TPO) by 6-propyl-2-thiouracil (PTU) and 1-methyl-2-mercaptoimidazole (MMI) used in the therapy of hyperthyroid patients was studied in vitro. The inhibition of TPO by MMI was not restored either by dialysis or by dilution, but the inhibition by PTU was restored by both treatments. PTU interacted directly with the product of TPO action (oxidized iodide) in the reaction mixture without significantly affecting TPO activity. MMI interacted directly with TPO and inhibited enzyme activity, rather than interacting with the product (oxidized iodide). The inhibition was irreversible with MMI, but reversible with PTU. The concentrations of PTU and MMI producing 50% inhibition of TPO were 2 x 10-6m and 8 x 10-7m, respectively, 2-Mercaptoimidazole inhibited TPO reversibly but 1-methylimidazole and imidazole did not. Both the methyl and mercaptoresidues in MMI moiety are thought to be essential to its irreversible inhibition of TPO. The in vivo effect of MMI and PTU on TPO activity was also studied. TPO activities in the thyroid homogenate of rats to which MMI (2 mg per rat) or PTU (10 mg per rat) had been administered intraperitoneally were determined before and after dialysis against buffer. TPO activity in the PTU treated thyroid homogenate was significantly lower than that in the control before dialysis, but the activity was restored to the control value after dialysis. On the contrary, TPO activity in the MMI treated thyroid homogenate was significantly lower than that in the control and was not affected by dialysis. These data may explain why MMI is a more potent inhibitor of iodination than PTU and may fit the clinical results observed when hyperthyroid patients are treated with these agents.