Abstract Fluorescence and circular dichroism spectral measurements, thermal denaturation studies and binding competition experiments with netropsin and actinomycin D were carried out in systems containing phenosafranine bound to DNA's differing in base composition. The investigated properties exhibit a heterogeneity related to the content of A . T and G . C pairs in DNA and to the nature of phenosafranine binding modes. At low level of saturation of binding sites ( r < 0.1) phenosafranine does not show strong preference for any of the DNA base pairs in the overall binding. However, the strong monomer non-cooperative binding outside the helix (mode I 1) occurs predominantly, even though not exclusively in G . C rich regions. The strong binding modes involving intercalated dye molecules (mode I 2 and eventually mode II 1) prevail in A . T rich regions. These binding modes become the principal types of strong phenosafranine interaction with DNA when the level of saturation of binding sites increases, i.e. at r > 0.1.