The transport of [14C]glycyl-glycine (Gly-Gly) across the tonoplast of isolated barley vacuoles has been characterized. Uptake of the dipeptide Gly-Gly into barley mesophyll vacuoles was strongly increased by the addition of ATP, while Mg2+ inhibited the ATP-dependent fluxes. Inhibition of the vacuolar proton pump by bafilomycin or dissipation of the delta pH had no effect on the ATP-dependent Gly-Gly uptake. Only the non-hydrolysable ATP analogue 5'-adenylylimidodiphosphate could partially substitute for ATP; ADP, GTP and UTP had no effect. Transport of Gly-Gly was saturable exhibiting an apparent Km of 49 +/- 5 mM. Uptake was inhibited by the sulphydryl reagents N-ethylmaleimide and p-chloromercuribenzenesulphonic acid. When present at 30 mM, various dipeptides as well as tri-glycine inhibited Gly-Gly (10 mM) uptake by 30-50%, whereas 5 mM phenylalanine was able to inhibit Gly-Gly uptake almost completely. Comparison with published data suggests that Gly-Gly is transferred across the tonoplast by the same system as several amino acids and inorganic ions.