The FAD binding site of rabbit liver glutathione reductase has been explored by reconstitution of the apoprotein with several FAD analogs modified in the isoalloxazine ring. The apoglutathione reductase binds the p-quinoid form of 8-mercapto-FAD, suggesting that the protein stabilizes a negative charge in the -N1-C2 = O position of the pyrimidine subnucleus. The main absorption peak in the visible spectrum of the 8-mercapto-FAD-enzyme is at 585 nm; treatment of the reconstituted protein with reducing agents of disulfide groups induces a reversible hypochromic shift of 20 nm of the peak. Thus, in 8-mercapto-FAD-glutathione reductase, the oxidation-reduction state of the active center disulfide can be monitored. The chemical reactivity toward methylmethanethiosulfonate and iodoacetamide of the 8-mercapto-FAD-enzyme shows that the flavin position 8 is freely accessible to solvent. However, position 2 is buried within the protein molecule as judged from the lack of reactivity of the 2-thio-FAD-enzyme with methylmethanethiosulfonate. Hydrogen peroxide reacts slowly with both 2-thio-FAD-enzyme and native glutathione reductase, yielding inactive enzyme with a modified spectrum; the prosthetic group is still protein bound. Differences in the active site of the rabbit liver enzyme compared to the human erythrocyte glutathione reductase are evidenced by use of FAD analogs: the peaks of reconstituted liver enzymes are shifted about 10 nm toward longer wavelengths.