Eukaryotic cells are divided into the nucleus and the cytosol, and, to enter the nucleus, proteins typically possess short signal sequences, known as nuclear localization signals (NLSs). Although NLSs have long been considered as features unique to eukaryotic proteins, we show here that similar or identical protein segments are present in ribosomal proteins from the Archaea. Specifically, the ribosomal proteins uL3, uL15, uL18, and uS12 possess NLS-type motifs that are conserved across all major branches of the Archaea, including the most ancient groups Microarchaeota and Diapherotrites, pointing to the ancient origin of NLS-type motifs in the Archaea. Furthermore, by using fluorescence microscopy, we show that the archaeal NLS-type motifs can functionally substitute eukaryotic NLSs and direct the transport of ribosomal proteins into the nuclei of human cells. Collectively, these findings illustrate that the origin of NLSs preceded the origin of the cell nucleus, suggesting that the initial function of NLSs was not related to intracellular trafficking, but possibly was to improve recognition of nucleic acids by cellular proteins. Overall, our study reveals rare evolutionary intermediates among archaeal cells that can help elucidate the sequence of events that led to the origin of the eukaryotic cell.,? The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: [email protected].