The process of selective nuclear protein transport is divided into at least two steps: 1) ATP-independent, nuclear localization signal (NLS)-dependent binding to the cytoplasmic face of nuclear pores and 2) ATP-dependent translocation through the nuclear pores. Using a digitonin-permeabilized cell-free transport assay, it was found that a karyophile forms a stable complex with a cytoplasmic fraction to target the nuclear pores. Since this complex shows nuclear porebinding activity, we have referred to it as the nuclear pore-targeting complex (PTAC). The complex contains two essential proteins. The 58 kDa component of PTAC (PTAC 58; importin α; karyopherin α) was found to bind directly to NLS. The 97 kDa component of PTAC (PTAC 97; importin β; karyopherin β) associates with PTAC 58, but not karyophile. A complex of PTAC 58 and PTAC 97 targets nuclear pores, depending on the presence of a karyophile. The data suggest that the initial step in nuclear protein transport occurs as a result of complex formation of a karyophile with PTAC 58 which is, in turn, bound to PTAC 97.