The sex-determining factor SRY plays an important role in male sexual development, diverting primordial gonads from the ovarian pathway toward male differentiation to form testes. SRY is a DNA-binding protein and gains access to the nucleus through two independently acting nuclear localization signals (NLSs) that flank the high mobility group (HMG) DNA-binding domain. We have reconstituted the nuclear import of SRY using an in vitro nuclear transport assay, showing that nuclear import of SRY can occur in the absence of additional exogenous cytosolic factors, with a significant reduction in nuclear transport in the presence of antibodies to the nuclear transport protein importin (Imp) beta1 but not Impalpha. We have also shown using in vitro binding assays that the C-terminal NLS of SRY binds directly to Impbeta1. Finally, we have shown that SRY can target green fluorescent protein to the nucleus in a mammalian transfected cell line; importantly, mutations known to result in sex reversal that map to either NLS impair nuclear accumulation implying that SRY nuclear import is critical to its function.