Complexes of 4’,6-diamidino-2-phenylindole (DAPI) with [poly(dG-dC)]2 and [poly(dI-dC)]2 were studied by optical spectroscopic techniques including linear dichroism (LD), circular dichroism (CD), and fluorescence measurements. The aim was to investigate the importance of the exocyclic amino group of guanine that protrudes into the minor groove of [poly(dG-dC)]2 but is absent in [poly(dI-dC)]2. When bound to [poly(dG-dC)]2, DAPI exhibits a negative, and strongly wavelength-dependent, reduced linear dichroism (LDr) in the DAPI absorption region, a weak positive CD, and a fluorescence behavior that is similar to that of free DAPI with accessibility to quenching by the aqueous solvent. These spectroscopic properties have been interpreted in terms of a major-groove binding geometry by Kim et al. (Kim, S. K.; Eriksson, S.; Kubista, M.; Norden, B. J. Am. Chem. Soc. 1993,115,3441-3447). By contrast, when bound to [poly(dI-dC)]2, DAPI exhibits a strong positive CD in the 300-420-nm region, a positive (wavelength-independent) LD(r), a strong increase of the fluorescence intensity, and shielding to added quencher. These spectroscopic properties closely resemble those of the DAPI-[poly(dA-dT)]2 complex, in which DAPI is situated deep in the minor groove of the polynucleotide. We conclude that the major groove binding geometry of DAPI when complexed with [poly(dG-dC)]2 is a result of a decreased affinity to the minor groove of [poly(dG-dC)]2 due to steric hindrance and decreased electronegative attraction caused by the amino group of guanine.