Despite the apparent single-stranded conformation and the absence of a GpC site, d(TGTCATTG) has been found to bind strongly to actinomycin D (ACTD) with 1:1 drug to strand binding stoichiometry. A hairpin binding model was speculated in which the planar phenoxazone chromophore inserts at the GTC site by pushing out the T-base while the terminal G folds back to form a G small middle dotC base pair so that the 3'-sides of both G-bases stack on the opposite faces of the phenoxazone plane. However, it was also suggested that a slipped duplex binding with similar binding principle could also be operative at higher DNA concentrations. To support such a contention, ACTD binding studies were made with d(TGTCATG) and related oligomers. This heptamer differs from the parent octamer d(TGTCATTG) by a mere removal of a T-base which should result in an enhancement of dimeric duplex formation and a concomitant reduction in monomeric hairpin contribution. It was found that ACTD binds well to d(TGTCATG) with 1 drug to 1 strand (or 2 drugs to 1 duplex) binding stoichiometry. These results are consistent with a slipped duplex binding model in which a dimeric duplex is formed at the self-complementary CATG tetranucleotide sequence with extruding TGT ends. Two drug molecules are bound at both ends of the duplex by pushing out the T-bases of GTC's so that the opposite faces of each phenoxazone are stacked by the 3'-sides of the two G-bases on opposite strands. Such a model provides a ready explanation for the observed enhancement in ACTD binding to d(TGTCATGTC) and d(TGTCATGTCA), where additional base pairs at the ends will stabilize GTC/GTC binding sites, and to d(TGTCAATTG) in which two additional base pairs facilitate the slipped-duplex formation. The observed ACTD affinity reductions for oligomers containing GTTC instead of GTC are also consistent with the T-base displacement model. These findings greatly expand the repertoire of ACTD binding to DNA and may have important implications on understanding the transcription inhibitory activities of this drug.