ABSTRACT The E6 oncoprotein of high-risk human papillomavirus type 16 (HPV16) interacts with several nuclear transcription factors and coactivators in addition to cytoplasmic proteins, suggesting that nuclear import of HPV16 E6 plays a role in the cellular transformation process. In this study we have investigated the nuclear import pathways of HPV16 E6 in digitonin-permeabilized HeLa cells. We found that HPV16 E6 interacted with the karyopherin (Kap) α2 adapter and could enter the nucleus via a classical Kap α2β1-mediated pathway. Interestingly, HPV16 E6 also interacted, via its basic nuclear localization signal (NLS) located at the C terminus, with both Kap β1 and Kap β2 import receptors. Binding of RanGTP to these Kap βs inhibited their interaction with HPV16 E6 NLS. In agreement with these binding data, nuclear import of the HPV16 E6 oncoprotein in digitonin-permeabilized HeLa cells could be mediated by either Kap β1 or Kap β2. Nuclear import via these pathways required RanGDP and was independent of GTP hydrolysis by Ran. Significantly, an E6 R124G mutant had reduced nuclear import activity, and the E6 deletion mutant lacking 121 KKQR 124 was not imported into the nucleus. The data reveal that the HPV16 E6 oncoprotein interacts via its C-terminal NLS with several karyopherins and exploits these interactions to enter the nucleus of host cells via multiple pathways.