(1) The inactivation of various forms of nucleoside transport with reagents blocking thiol groups was studied in whole cells of E. coli B. No positive correlation between the efficiency of active transport and the extent or rate of inactivation could be demonstrated. (2) The most efficient constitutive nucleoside-transporting system was found to comprise a specific thiol component characterized by low rate of inactivation with N-ethylmaleimide; the less efficient inducible transport and the facilitated diffusion of guanosine require the integrity of another thiol component which is rapidly inactivated with N-ethylmaleimide. (3) The constitutive nucleoside-transporting system is completely inactivated with T4 phage, while other modes of nucleoside transport are much less affected. (4) Inactivation of constitutive transporting system in cells exposed to N-ethylmaleimide for a limited period of time continues long after the inhibitor has been removed, indicating storage of the inhibitor in some cellular compartment. Addition of dithiothreitol stops the inactivation immediately.