Mithramycin (MTR), an aureolic acid group of antitumor antibiotic is used for the treatment of several types of tumors. We have reported here the association of MTR with an essential micronutrient, manganese (Mn(2+)). Spectroscopic methods have been used to characterize and understand the kinetics and mechanism of complex formation between them. MTR forms a single type of complex with Mn(2+) in the mole ratio of 2:1 [MTR: Mn(2+)] via a two step kinetic process. Circular dichroism (CD) spectroscopic study indicates that the complex [(MTR)2 Mn(2+)] has a right-handed twist conformation similar in structure with the complexes reported for Mg(2+) and Zn(2+). This conformation allows binding via minor groove of DNA with (G, C) base preference during the interaction with double-stranded B-DNA. Using absorbance, fluorescence, and CD spectroscopy we have shown that [(MTR)2 Mn(2+)] complex binds to double-stranded DNA with an apparent dissociation constant of 32 μM and binding site size of 0.2 (drug/nucleotide). It binds to chicken liver chromatin with apparent dissociation constant value 298 μM. Presence of histone proteins in chromatin inhibits the accessibility of the complex for chromosomal DNA. We have also shown that MTR binds to Mn(2+) containing metalloenzyme manganese superoxide dismutase from Escherichia coli.