Recognition of sequences within duplex DNA is a general strategy for probing DNA function and for disrupting gene expression. Peptide nucleic acids (PNAs) and conjugates between DNA oligonucleotides and cationic peptides possess superior potential for strand invasion at complementary sequences. To elucidate the rules underlying this phenomenon we examined hybridization to sequences throughout plasmid pUC19. We discovered that oligonucleotide-peptide conjugates and PNAs fall into three classes based on their hybridization efficiencies: (i) those complementary to inverted repeats within AT-rich region hybridize with highest efficiency; (ii) those complementary to areas adjacent to inverted repeats or near AT-rich regions hybridize with moderate efficiency; and (iii) those complementary to other regions do not detectably hybridize, with the exception of PNAs that have been modified to incorporate additional;positive charge. Hybridization of oligonucleotide-peptide conjugates and PNAs was stringently dependent on target sequence and was most efficient at sequences within the promoter for beta-lactamase or prior to the Escherichia coli origin of replication, suggesting that the sequences that regulate biological function may also be among the most susceptible to strand invasion. The correlations between oligomer chemistry, DNA target sequence, and hybridization efficiency that we report here have important implications for the recognition of duplex DNA in cell-free systems and for the selection of target sites for regulating gene expression within cells using synthetic molecules.