Ethidium was found to be efficiently cross-linked to DNA by glyoxal. Kinetic studies showed that the rate of the cross-linking reaction is strongly dependent on the glyoxal concentration. Comparative studies using a series of phenanthridines and acridines showed that NH2 groups at both the 2 and 7 positions on the phenanthridine ring are necessary for efficient cross- linking. Studies using synthetic polydeoxynucleotides showed that the 2- amino group of guanine is absolutely required for cross-linking. Fluorescence contact energy transfer and relative viscosity experiments showed that the cross-linked drug remains intercalated into DNA. DNA gel electrophoresis and melting studies demonstrated that cross-linked ethidium does not dissociate the DNA double helix to single strands.