The binding of tRNA species from calf liver to immobilized exportin-t in the presence of ran x GppNHp was examined by affinity chromatography. We observed different eluting behaviors of individual tRNAs. After separating tRNAs on a two-dimensional polyacrylamide gel, the positions of seven selected tRNAs were identified by Northern hybridization and their relative affinities to immobilized exportin-t x ran x GppNHp complex were estimated in the order of tRNA(Leu)(CAG) > tRNA(Ser)(GCU), tRNA(Leu)(CAA), tRNA(Ser)(UGA) > tRNA(Ser)(AGA), tRNA(Leu)(AAG) > tRNA(Arg)(ACG). We propose that exportin-t preferentially binds and exports those tRNAs that are rapidly consumed by the protein synthesis machinery.