The reaction of yeast hexokinase with iodoacetate or iodoacetamide has been investigated in detail, using pure hexodinase B. Of the four thiols in each subunit of the molecule, two (the "apparently essential thiols") are alkylated rapidly at 35 degrees, and the enzymic activity is lost in parallel with their reaction. The other two thiols react subsequently to completion, but at a very much slower rate. In the conditions use, no other uptake of the reagent occurs elsewhere during these thiol alkylations. Electrophoretically homogeneous kialkylated and tetraalkylated protein species are formed, in the two stages of the reaction. The inactivating reaction at 35 degrees with the apparently essential thiols is second order. The rate constant increases with increasing pH, in the range pH 7.0-8.5, in a manner consistent with control of the reaction by a group with pKa of approximately 10. The absolute (pH independent) rate constant is of the same order as that for a normal thiol in model compounds. The availability of the apparently essential thiols appears to be associated with some conformational change in the molecule in the monomer form: it declines at high ionic strengths, is maximal at intermediate values where the dimer first dissociates, but is lowered in the dimer at very low ionic strengths. The reaction also shows a sharp temperature dependence: the dimer at 30 degrees (in constrast to 35 degrees) shows no availability of the apparently essential thiols. A similar transition to a state permitting fast inactivation is found with pH, above pH 8.5. The reaction of the two apparently essential thiols is strongly inhibited by glucose. ATP and ADP, and their Mg complexes, protect significantly, but less effectively than does glucose. The affinities of these substrates at the active site of the enzyme are measured in this protection system. These various reactions appear to be of value for identifying the cysteine-containing regions that are involved in the active center or in its maintenance in the structure.