Morpholino and phosphorothioate (S-DNA) antisense oligos were compared in both cell-free and in-cell translation systems. In the most stringent test of specificity in the cell-free system, a globin-targeted S-DNA oligo was found to inhibit its target sequence at concentrations of 10 nM and above, but the sequence-specific component of this inhibition dropped below 50% at concentrations of 100 nM and above. A corresponding Morpholino oligo achieved even higher inhibition at 10 nM, but in contrast to the S-DNA, with the Morpholino, the sequence-specific component of this inhibition remained above 93% at a concentration of 3000 nM. In this same cell-free test system, several S-DNA oligos exhibited substantial undesired nonantisense effects at concentrations of 300 nM and above, whereas corresponding Morpholino oligos exhibited little or no nonantisense activity through a concentration of 3000 nM. In scrape-loaded HeLa cells, both globin-targeted and HBV-targeted S-DNAs (both antisense and control oligos) generally failed to achieve significant translational inhibition at extracellular concentrations up to 3000 nM. In contrast, the Morpholino oligos achieved effective and specific translational inhibition at extracellular concentrations ranging from 30 nM to 3000 nM.