Summary1. Natural selection can generate correlated suites of phenotypic traits by acting independently on physiological and behavioural characters or on mechanisms that exert pleiotropic actions. 2. Current theory, supported by artificial selection studies, suggests that physiological and behavioural stress responses are at least partially under genetic control and covary in a predictable manner. Indeed, physiological mechanisms such as hormonal stress responsiveness may underlie variation in behaviour, including consistent behaviours described as temperament or personality, with bolder, more exploratory and active individuals being less hormonally responsive to stressors. 3. This relationship, however, has yet to be demonstrated in natural populations. We investigated the relationship between hormonal and behavioural stress responsiveness in multiple natural populations of a tropical freshwater poeciliid fish, Brachyrhaphis episcopi, that experience different levels of predation pressure and hence encounter different rates of stressful events. Predation can impose a strong selection pressure, and living with a high risk of predation is known to select for specific behavioural traits. 4. We quantified variation in stress responsiveness via cortisol release rates (exp. 1) and behaviour in an open field test followed by cortisol release rates (exp. 2). Populations exposed to high levels of predation were consistently more exploratory and active and had lower release rates of cortisol in response to a stressor than conspecifics sampled at sites with few predators. 5. However, this difference in stress responsiveness was only apparent after fish had experienced the mild stress of behaviour testing (in exp. 2), which resulted in elevation of cortisol levels. The relationship between hormone release and behaviour was also not apparent within populations once independent factors were controlled for, highlighting the importance of factors such as size and sex on individual variability. 6. This study demonstrates that the relationship between hormonal and behavioural stress responsiveness can result from natural selection pressures, such as that imposed by predation.