The antisense activity of oligomers with 2′-O-methyl (2′-O-Me) phosphorothioate, 2′-O-methoxyethyl (2′‐O-MOE) phosphorothioate, morpholino and peptide nucleic acid (PNA) backbones was investigated using a splicing assay in which the modified oligonucleotides blocked aberrant and restored correct splicing of modified enhanced green fluorescent protein (EGFP) precursor to mRNA (pre-mRNA), generating properly translated EGFP. In this approach, antisense activity of each oligomer was directly proportional to up-regulation of the EGFP reporter. This provided a positive, quantitative readout for sequence-specific antisense effects of the oligomers in the nuclei of individual cells. Nuclear localization of fluorescent labeled oligomers confirmed validity of the functional assay. The results showed that the free uptake and the antisense efficacy of neutral morpholino derivatives and cationic PNA were much higher than that of negatively charged 2′-O-Me and 2′-O-MOE congeners. The effects of the PNA oligomers were observed to be dependent on the number of l-lysine (Lys) residues at the C-terminus. The experiments suggest that the PNA containing Lys was taken up by a mechanism similar to that of cell-penetrating homeodomain proteins and that the Lys tail enhanced intracellular accumulation of PNA oligomer without affecting its ability to reach and hybridize to the target sequence.