The effect of the number and position of the positive charges on porphyrin with respect to the mode of binding to poly[d(G-C)2] and poly[d(A-T)2] were investigated by absorption and polarized spectroscopy, including circular and linear dichroism (CD and LD). Meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP), which possesses four positive charges on the periphery pyridinium rings, produces a negative CD and wavelength-independent reduced LD (LDr) spectra in the Soret absorption region when it associates with poly[d(G-C)2]. These spectral characteristics have been considered as diagnostic for intercalation. In contrast, both trans- and cis-bis(N-methylpyridinium-4-yl)diphenylporphyrin (trans- and cis-BMPyP), where the number of positive charges was reduced to two, multisignate CD and strong wavelength-dependence of the LDr spectra were observed, indicating that these porphyrins do not intercalate. Therefore, four positive charges are required for TMPyP intercalation. When associated with poly[d(A-T)2], trans-BMPyP exhibited a positive CD signal at a low [porphyrin]/[nucleobase] ratio with the appearance of a bisignate CD upon increase of the mixing ratio, suggestive of binding at the groove of the double helix at low mixing ratios, and stacking at increasing mixing ratios. Conversely, no monomeric binding was evident in the bis-BMPyP bisignate CD spectrum; hence, only the stacking mode was found for cis-BMPyP, even at the lowest [porphyrin]/[nucleobase] ratio, suggesting the importance of the position of the positive charges in determining monomeric groove binding or stacking. The binding geometries of trans- and cis-BMPyP were similar when associated with poly[d(A-T)2], as determined from the similar LDr spectrum. When associated with DNA, TMPyP exhibited similar spectral properties with that of the TMPyP-poly[d(G-C)2] complex, indicating intercalation of TMPyP between the DNA base pairs. Conversely, CD and LDr characteristics of both trans- and cis-BMPyP-DNA complexes resembled those that complexed with poly[d(A-T)2] at a high [porphyrin]/[DNA] ratio, suggesting that both porphyrins were stacked along the DNA stem.