Fluorescent behaviour of lomustine, a DNA cross-linking agent, was investigated in different solvents, pH and in the presence of b-cyclodextrin (b-CD). The solvents in which fluorescence spectra were observed play a major role in determining the spectral intensity of fluorophore, since it was found to exhibit new fluorescent properties essentially influenced by intermolecular interactions, particularly by intermolecular H-bonding formed with solvents. The pH-dependence profile was typically U-shape with a maximum at pH between 3.51 and 6.58. It was corroborated that the fluorescence emission band of lomustine is significantly intensified in the presence of b-CD. From the changes in the fluorescence spectra, it was concluded that b-CD forms a 1:1 inclusion complex with lomustine and its association constant was calculated.