One of the major barriers to the development of antisense therapeutics has been their poor bioavailability. Numerous oligonucleotide modifications have been synthesized and evaluated for enhanced cellular permeation with limited success. Phenoxazine, a tricyclic 2' deoxycytidine analog, was designed to improve stacking interactions between heterocycles of oligonucleotide/RNA hybrids and to enhance cellular uptake. However, the bioactivity and cellular permeation properties of phenoxazine-modified oligonucleotides were unknown. Incorporation of four phenoxazine bases into a previously optimized C-5 propyne pyrimidine modified 7-mer phosphorothioate oligonucleotide targeting SV40 large T antigen enhanced in vitro binding affinity for its RNA target and redirected RNAse H-mediated cleavage as compared with the 7-mer C-5 propynyl phosphorothioate oligonucleotide (S-ON). The phenoxazine/C-5 propynyl U 7-mer S-ON showed dose-dependent, sequence-specific, and target-selective antisense activity following microinjection into cells. Incubation of the phenoxazine/C-5 propynyl U S-ON with a variety of tissue culture cells, in the absence of any cationic lipid, revealed unaided cellular penetration, nuclear accumulation, and subsequent antisense activity. The unique permeation properties and gene-specific antisense activity of the 7-mer phenoxazine/C-5 propynyl U S-ON paves the way for developing potent, cost-effective, self-permeable antisense therapeutics.