ABSTRACT Biofluorescence, the ability to exploit light with short wavelengths, such as ultraviolet (UV) and blue, and re-emit it with lower energy and longer wavelengths, is considered a widespread phenomenon among marine animals and has many ecological roles in intra- and interspecific communication. Heterobranch sea slugs are believed to rely almost exclusively on their colour patterns for survival, with coloration serving as camouflage or as a warning to predators of heterobranch toxicity (aposematic coloration). Since solar light is progressively absorbed by seawater with a constant loss of longer wavelengths, it is unclear how heterobranch coloration can maintain its ecological role below the water surface. The present study investigates, for the first time, in situ biofluorescence in 53 species of Mediterranean Heterobranchia to determine whether these animals rely on light emission to enhance the visibility of their colour patterns. The ability to fluoresce when illuminated by UV and blue light (wavelengths <470 nm) appeared to be quite widespread among nudibranchs (i.e. found in c. one-third of the encountered species), while no evidence of light emission was found in non-nudibranch heterobranchs. The majority of fluorescent species were Cladobranchia belonging to the superfamilies Fionoidea and Aeolidioidea. Nudibranchs fluoresced in four different colours, with green being the most common. Light emission was often restricted to particular body regions, such as the cerata of Cladobranchia. These preliminary data might indicate that fluorescence plays an essential role in the enhancement of nudibranch aposematic coloration.