We have been developing a gene silencing technology by harnessing a tRNA 3' processing endoribonuclease, tRNase Z, with antisense oligonucleotides. Here, to further improve this technology, we investigated how the length and the modifications of naked oligonucleotides affect the efficiency of their uptake by HeLa, HEK293, and HL60 cells by flow cytometry and fluorescence microscopy. 7-30-nt Alexa-Fluor-568-labeled DNAs with phosphorothioate linkages and 7-30-nt Alexa-Fluor-568-labeled, 2'-O-methylated RNAs without phosphorothioate linkages were examined, and, on the whole, longer oligonucleotides were shown to be intracellularly taken up more efficiently. In addition, a 2'-O-methoxyethylated RNA without phosphorothioate linkages, a 2'-fluoriated RNA without phosphorothioate linkages, a 2'-O-methylated RNA with phosphorothioate linkages, and a 2'-O-methylated RNA with phosphorothioate linkages and LNA modifications of 5'-/3'-terminal nucleotides were examined. The oligonucleotides with phosphorothioate linkages were taken up by the cells more efficiently than those without the linkages. Furthermore, we examined how the phosphorothioate linkages of oligonucleotides affect their antisense effects using 22-nt anti-miR16 oligonucleotides with and without phosphorothioate linkages. The latter oligonucleotide decreased the miR16 level much more intensively than the former, although the latter was intracellularly taken up much less efficiently. These observations may be not generalized and differ depending on features of oligonucleotides and cell types. Taken together these results suggest that the productive uptake efficiency for an antisense oligonucleotide needs to be considered to select its length and modifications.