Human disturbance is thought to be a major source of stress for animals but breeding status, social interactions and food availability are also potential sources. Long-lasting stress may adversely affect the fitness of animals and for that reason the evaluation of stressors is important for conservation of threatened species. The aim of our study was therefore to assess which factors cause stress in wolves (Canis lupus). We evaluated the stress levels of wolves from six packs by measuring the concentration of glucocorticoid metabolites in 59 faecal samples with a Cortisol enzyme-immunoassay. During the breeding season, stress hormone concentration was higher than during the rest of the year, with two peaks around mating and begin of denning, respectively. Multiple regressions ranked by AIC showed that breeding had the highest impact on the wolves' stress levels, followed by human activity, pack size, and prey density. We conclude that human activity is only one of several factors contributing to stress in wolves and that intraspecific competition during breeding is likely to cause elevated levels of glucocorticoids.