The crystal and molecular structure of ethidium monoazide, a photoactive derivative of ethidium, was determined from X-ray diffraction data. Crystals of 8-azidoethidium chloride monohydrate are monoclinic, space group P21/c, with a = 8.336(5), b = 14.778(6), c = 16.873(5) Å, and β = 99.90(3) ° The crystals are poorly ordered, weakly diffracting, and unstable in air. Intensity data were collected with an automated diffractometer from a crystal that was sealed in a thin-walled glass capillary. The structure was solved by direct methods and was refined by least squares to R = 0.20 for the complete set of 2126 unique reflections, and to R = 0.11 for 724 reflections with 1 > 2σ(I). The results demonstrate that the photoactive azide moiety is attached to the 8-position of the phenanthridinium moiety. This azide group is almost linear, is nearly in the plane of the phenanthridinium ring, and is oriented away from the phenyl substituent (i.e., trans to the C(7)-C(8) bond). Possible models for intercalation complexes of 8-azidoethidium with double-helical nucleic acids were examined by modifying published coordinates from crystal structures of ethidium-dinucleotide complexes. When in the conformation observed in this crystal structure, 8-azidoethidium can be substituted for ethidium in the published complexes without suffering any unacceptable steric interactions. © 1979 Plenum Publishing Corporation.