Recently, we described a new class of antisense oligonucleotides that can be used to direct the cleavage of mRNAs in Xenopus laevis embryos by RNase H (Dagle et al., Nucleic Acids Res. 18, 4751-4757). In this study, we have examined several factors that determine the activity of these derivatives. In embryos, oligodeoxyribonucleotides were found to be rapidly degraded by a 3' exonuclease. Modification of 3'-terminal phosphodiester linkages as phosphoramidates blocks this activity. The predominant sites of endonucleolytic cleavage within the embryo are localized close to the 5' termini demonstrating the necessity of multiply modifying phosphodiester linkages at each end of the molecule. A stretch of at least six consecutive phosphodiester linkages is required to form an effective substrate for Xenopus RNase H; mRNA degradation with an oligonucleotide containing fewer than six contiguous unmodified internucleoside linkages is greatly diminished. Injection of an anti-cyclin B oligonucleotide containing eight unmodified residues results in degradation of cyclin B mRNAs and subsequent inhibition of embryonic cell division. An oligonucleotide with the same sequence but containing four consecutive phosphodiesters has no observable effect on the cell cycle. This last observation suggests that, in Xenopus embryos, hybridization alone has a limited role, if any, in oligonucleotide-mediated inhibition of gene expression.