Liver cytosolic gamma-cystathionase catalyzes the generation of reduced sulfur species, referred to as "bound sulfur,' in the presence of cystine. Incubating a rat liver cytosol fraction in the presence of cystine or oxidized glutathione inactivated certain cytosolic enzyme activities. The activities of cytosolic phosphofructokinase (PFK) and pyruvate kinase rapidly decreased at pH 7.4 during incubation with a lower concentration of cystine than during incubation with oxidized glutathione. Hexokinase and 11 other enzymes in the system were affected minimally or not at all. Adding dithiothreitol to the system reactivated the modified enzymes. Inactivated PFK activity could also be recovered when reduced glutathione or NADPH was added to the cytosol fraction. In these reconstitution systems, purified rat liver PFK was directly inactivated with cystine trisulfide (one of the low molecular types of bound sulfur), but not by cystine (below 0.1 mM). Purified PFK was also inactivated by incubation with cystine plus gamma-cystathionase freshly prepared from cytosol. This was not observed, however, when gamma-cystathionase was pretreated with a specific inhibitor, D,L-propargylglycine. The cystine-dependent inactivation of PFK observed in liver cytosol is shown to be caused mainly by the reaction between bound sulfur and the enzyme, but not by the direct thiol/disulfide exchange. Thus, in vitro modification of the cytosolic enzymes by bound sulfur generated from cystine with gamma-cystathionase has high potency and relatively specific.