Comparative electrophoretic, thermal denaturation, and spectroscopic studies with dodecamers of the form d(ATTA-XGCX-TAAT) and their self-complementary counterparts suggest that actinomycin D (ACTD) binds strongly to a 5'GC3' site with flanking T/T mismatches and moderately to that with C/C mismatches but weakly to those with other G/G or A/A mismatches. The relative binding order is found to be T/T > C/C > G/G > A/A. The ACTD binding affinity for the GC site with T/T mismatches is comparable to the strong binding of self-complementary-XGCY-sequences. Both the ACTD association and dissociation kinetics at the GC site with flanking T/T mismatches require two-exponential fits. The slow component of the association rates is slower than those of the self-complementary sequences, whereas that of the dissociation is only slightly faster than that of the -TGCA- sequence. Interestingly, the slow component of dissociation is decidedly slower than those of -AGCT- and -CGCG- sites and is more than an order of magnitude slower than those with C/C, G/G, and A/A mismatches. These kinetic results are further corroborated by fluorescence measurements using 7-amino-ACTD, a fluorescent analog of ACTD. In addition, fluorescence and absorbance spectral characteristics indicate that the binding mode at the GC site with flanking T/T mismatches resembles those of strong-binding self-complementary -XGCY- sites which are known to be intercalative in nature. The observed slow ACTD dissociation at the T/T-mismatched site suggests that the minor-groove environment near the T/T-mismatched pairs provides favorable interactions with the pentapeptide rings of the drug, whereas the others, especially those of bulkier purine/purine mismatches, result in less favorable interactions.