Nuclear transport is mediated by transport factors, including the importin beta family members. The directionality of nuclear transport is governed by the asymmetrical distribution of the small GTPase Ran. Of note, importin alpha/beta-mediated import of classical nuclear localization signal (cNLS)--containing cargo is more efficient than other Ran-dependent import pathways that do not require importin alpha. In this study, we characterized the role of importin alpha in nuclear transport by examining import efficiencies of cNLS-cargo/importin alpha/beta complexes. We first depleted digitonin-permeabilized semi-intact cells of endogenous importin alpha and used the cells to show that the interaction between importin alpha and Nup153--a component of the nuclear pore complex (NPC)--is essential for efficient import of importin beta-binding domain containing substrates, but not other cargoes that directly bind to importin beta. Moreover, we found that the binding of importin alpha to Nup153 facilitates cNLS-mediated import, and demonstrated that importin alpha in import complexes and cargo-free importin alpha prebound to Nup153 promote efficient import of cNLS-containing proteins. This is the first in vitro study showing that in conjunction with Nup153, importin alpha contributes to directionally biased exit of cNLS-containing cargo to the nuclear side of NPCs.