The antitumor antibiotic Altromycin H was studied using electronic absorption (UV-Vis.) and circular dichroism (CD) spectroscopy. The dissociation constants of the phenolic groups on C(5) and C(11) were estimated as pK(1)=6.7 and pK(2)=11.8 at 25 degrees C, respectively, and a complete assignment of the CD and UV-Vis. bands is proposed. The interaction of Cu(II) ions with the Altromycin H has been also investigated by UV-Vis., CD and electron paramagnetic resonance (EPR) spectroscopy. A pH depended stepwise complex formation was observed. At pH<4 no copper-Altromycin H interactions were detected. At the 4<pH<8 the Cu(II) ions coordinate to Altromycin H via the C(4)O and C(5)OH groups atoms of the chromophore ring and the [Cu(AltroH)(2)] complex (formation constant, K(f), 4.00+/-0.9 x 10(11) M(-2) at 25 degrees C) is the predominant species. In alkaline solution the hydroxo-bridged complex, [Cu(OH)(AltroH)](2), is proposed to be as the unique present species.