Luminescence assays are generally based on measurements of light intensity alone. Inclusion of colour as an additional parameter of the assay could increase the information content. Colour variation in luminescence is particularly prevalent among beetle luciferases. To study the relationship between enzyme structure and colour, luciferases from a Jamalcan click beetle were examined as a model system. These luciferases emit light ranging from green t o orange, though their amino acid sequences differ by less than 5%. Through mutation of their respective cDNA clones, the amino acids responsible for the colour variation were identified. These specific amino acids are few, and they act upon colour independently w i t h respect t o the enzyme structure. Analysis of their effects indicates that the potential for colour variation among beetle luciferases is greater than is evident among the click beetle luciferase. Because of the subtle changes of enzyme structure that effect colour, luciferases that emit different colours may be useful as paired genetic reporters. They should interact equivalently w i t h the intracellular environment of a host, but could be distinguished by colour in their assay. Such paired reporters could be used t o observed simultaneous events, or t o provide internal control for luminescence measurements.