Conjugation reaction of methazolamide with glutathione and its subsequent reactions were studied in vitro. Glutathione, cysteinylglycine, and cysteine conjugates of methazolamide were chemically synthesized. All of the three compounds showed absorbance below 330 nm, with maximal absorbance at approximately 300 nm. At the wavelengths below 220 nm, absorbance was proportional to the number of the amino acids each compound had. Amino acid analysis of the glutathione conjugate showed that the conjugation reaction involved the cysteine residue of glutathione. In order to identify the chemical structure of the reaction product, cysteine conjugate was subjected to infrared, proton nuclear magnetic resonance, and mass spectral analyses. These studies indicated that the cysteine conjugate was S-(5-acetylimino-4-methyl-delta 2-1,3,4-thiadiazolinyl)cysteine. The reaction with glutathione was not catalyzed by glutathione S-transferases, but proceeded in the absence of the enzyme. The glutathione conjugate was degraded by bovine ciliary body homogenate to the cysteinylglycine conjugate and then to the cysteine conjugate.