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The effects of acute (200 mg/kg) and chronic (20 mg/kg per day for 7 days) administration of acetazolamide on iodide transport, electrolyte distribution, and on carbonic anhydrase (CA), Na⁺,K⁺-ATPase and HCO₃^–-ATPase activities were evaluated in mouse thyroid glands. The effects of withdrawal from chronic administration of acetazolamide were also assessed. A single injection of a large dose of acetazolamide increased iodide uptake and completely inhibited CA activity. Chronic administration of acetazolamide only slightly increased iodide uptake; CA inhibition was also less marked than after acute administration. After withdrawal of acetazolamide, iodide uptake decreased and CA activity recovered rapidly to the control levels. Chronic treatment with acetazolamide decreased the content of water and increased the contents of protein and DNA in thyroid tissues. Withdrawal of the drug resulted in an increase in Na⁺ and K⁺ contents and a decrease in water content of this gland. These data demonstrate that CA activity has an inverse relation to the iodide transport and a direct relation to Cl^– content in the thyroid. Chronic administration of acetazolamide also increased iodide uptake by the thyroid glands of hypophysectomized rats and of turtles.