Carotenoids produce most of the brilliant orange and yellow colours seen in animals, but animals cannot synthesize these pigments and must rely on dietary sources. The idea that carotenoids make good signals because they are a scarce limiting resource was proposed two decades ago and has become the leading hypothesis for the role of carotenoids in animal communication. To our knowledge, until now, however, there has been no direct evidence that carotenoids are a limiting resource in nature. We showed that carotenoid availability in the wild limits the expression of sexual coloration in guppies (Poecilia reticulata), a species in which females prefer males with brighter orange carotenoid–containing spots. Further, the degree of carotenoid limitation varies geographically along a replicated environmental gradient (rainforest canopy cover), which opens new avenues for testing signal evolution theory.