The creation of null mutants by inhibiting the expression of specific genes using oligodeoxyribonucleotides (ODNs) may be an important tool in understanding early development. One strategy for altering gene expression involves inhibiting transcription by forming triplex DNA at important regulatory sites in the genome. We have previously shown that positively charged ODNs are able to specifically bind to DNA duplexes to form triplex DNA in vitro with Kd values in the nanomolar range. Using completely modified cationic ODNs targeted against the promoter region of the Xenopus laevis GS17 gene (one of the first transcripts produced in the embryonic frog) we have shown specific reduction in GS17 mRNA. Following the microinjection of 25 ng of modified ODN into single cell embyros, the steady state level of GS17 mRNA after 9 hours was reduced to approximately 50% of that seen in control embryos. Injection of ODNs targeting either of two triplex binding sites in the 5′ upstream regulatory region of the GS17 gene resulted in decreased mRNA levels. A second ODN based strategy for preventing gene expression is to accelerate the degradation of a specific mRNA prior to translation by forming DNA:RNA duplexes which are substrates for RNase H. Substrate recognition constraints inherent in this strategy require the presence of several unmodified phosphodiester linkages in the ODN. We have previously shown that partially modified ODNs with neutral phosphate modifications at each terminus can effectively direct degradation of specific mRNAs in Xenopus embryos. ODNs containing positively charged terminal modifications have proven to be even more effective at directing specific mRNA degradation in both oocytes and embryos. In Xenopus oocytes, an ODN containing 6 unmodified diester linkages flanked by neutral linkages was ineffective in reducing levels of AN2 mRNA. An ODN of the same sequence containing 6 unmodified linkages but flanked by cationic linkages reduced the level of AN2 mRNA to nearly undetectable levels within 3 hours. In Xenopus embryos both ODN constructs can direct mRNA degradation, however the positively charged ODN is more effective. The use of cationic ODNs to alter gene expression may prove useful for creating null mutations in certain vertebrate models in which conventional genetic manipulation is either impractical or impossible.