Objectives. To investigate whether the single-item fatigue question of the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and the Multidimensional Fatigue Inventory (MFI) are appropriate instruments to measure fatigue in ankylosing spondylitis (AS); to identify factors that influence fatigue in AS; and to assess how fatigue in all its aspects is associated with quality of life in AS. Methods. A total of 812 patients with AS were included. Patients completed questionnaires on disease activity (BASDAI), functional ability (Bath Ankylosing Spondylitis Functional Index [BASFI]), global well-being (Bath Ankylosing Spondylitis Global Score [BAS-G]), overall perceived health (EuroQoL visual analog scale), and quality of life (Ankylosing Spondylitis Quality of Life questionnaire, and Short Form 36 [SF-36]). Patients were dichotomized into a F+ group (fatigue = major symptom) if the BASDAI fatigue score was > 5.0 and a F- group (fatigue = minor symptom) if the fatigue score was < 5.0. Reproducibility was assessed with intraclass correlation coefficients, and responsiveness was calculated according to 3 different methods. Logistic regression analysis was used to determine which factors were associated with fatigue. Multiple regression analysis was used to investigate whether fatigue contributes in explaining quality of life. Results. Fifty-three percent of the patients were assigned to the F+ group. They scored significantly worse compared with the F- group with respect to each dimension of the MFI and to all other questionnaires studied (all P < 0.001). The BASDAI fatigue question, as well as each separate dimension of the MFI, showed moderate to good reproducibility (0.57-0.75) and responsiveness (0.23-0.96). Fatigue was, in the opinion of the patients, highly associated with pain (70% of patients) and stiffness (54% of patients). Logistic regression analysis showed that scores on BASDAI, BASFI, BAS-G, and mental health status (SF-36) were independently associated with fatigue (R2 = 0.52). Multiple regression analysis showed that scores on the BASDAI fatigue question were significantly associated with quality of life. With the 5 MFI dimensions as explanatory variables, different aspects of fatigue were associated with different domains of quality of life. Conclusion. Fatigue as a major symptom of AS can effectively be measured with either a single-item question on the intensity of fatigue or with the MFI. The MFI, however, provides more insight into specific dimensions of fatigue. Fatigue appears to be associated with the level of disease activity, functional ability, global well-being, and mental health status. In addition, fatigue negatively influences different aspects of quality of life.