The inhibition of gene transcription mediated by peptide nucleic acids (PNAs) has been described as mainly due to a process that involves strand invasion of target DNA containing homopurine stretches with generation of PNA/DNA/PNA triplexes. Scarce information is available on DNA strand invasion of mixed purine-pyrimidine stretches and theirs possible use as molecules able to interact with transcription factors. With this aim, we compared the effects of DNA/DNA, DNA/PNA or PNA/PNA hybrid molecules mimicking the NF-kappaB binding sites of HIV-1 on LTR-driven in vitro transcription. The results allowed us to conclude that PNA/PNA molecules and DNA/PNA hybrids are capable of inhibiting transcription, like the DNA/DNA. We investigated the mechanisms by which PNA/PNA and DNA/PNA hybrids affect the transcription. The results suggest that DNA/PNA and PNA/PNA molecules inhibit transcription in distinct manner; the inhibitory effects are due to strand invasion in the case of PNA/PNA hybrids and decoy effects on transcription factors belonging the NF-kappaB/Rel family in the case of DNA/PNA hybrids. It is noteworthy that DNA/PNA hybrids are more resistant to nucleases than DNA/DNA hybrids.