Using thiol blocking agents, we examined the role of sulphydryl groups for function of the lysosomal sulphate transport system. Monothiol binding reagents, p-hydroxymercuribenzoic acid (p-HMB) and p-chloromercuribenzene sulphonic acid (p-CMBS), dithiol binding reagents such as CuCl2, the alkylating agent, N-ethylmaleimide (NEM), and NADH all inhibited lysosomal sulphate transport. The inhibitory effects of NEM and Cu2+ were not additive, suggesting that they both act upon the same critical sulphydryl group(s). Unlike the case for NEM, the inhibitory effects of Cu2+ were reversed by the reducing agent, dithiothreitol. Exposure to NEM resulted in a seven-fold increase in Km to 867 microM versus a control value of 126 microM and a modest decrease in Vmax to 99 pmolperunit beta-hexosaminidase per 30 s versus a control value of 129 pmolperunit beta-hexosaminidase per 30 s. Similar although somewhat less dramatic results were obtained using Cu2+ with an increase of Km to 448 microM and a Vmax of 77 pmolperunit beta-hexosaminidase per 30 s. The sulphate transport activity of detergent solubilized lysosomal membranes could be bound to a p-chloromercuribenzoic acid (p-CMB)-Sepharose sulphydryl affinity resin and eluted with mercaptoethanol. Sulphydryl groups thus appear to play a role in sulphate transport through effects on substrate affinity. Sulphydryl-binding appears to be a strategy that may be useful for purification of the transporter.