Psychological flexibility is the act of being open to internal experiences while pursuing valued life directions and has been implicated in positive mental health. A lack of psychological flexibility has been implicated in a wide range of mental health problems. In most research, assessment of psychological (in)flexibility has been done with the Acceptance and Action Questionnaire – II (AAQ-II), yet researchers have noted that items on the AAQ-II may not adequately discriminate between responses to experiences and the experiences themselves. Furthermore, little research has examined whether items on the AAQ-II function as intended in terms of assessing psychological (in)flexibility, and whether items function differently across populations. The present study used an item response theory framework to examine item functioning in the AAQ-II across items (within the measure) and across non-distressed student, distressed student, outpatient, and residential samples. The analyses identified differences in functioning between items, with some items being more sensitive to differences in psychological inflexibility. No items performed well in assessing psychological flexibility (as opposed to inflexibility) or positive functioning. Items functioned similarly across samples, yet patterns of responding differed in the non-distressed student versus residential and outpatient samples. Implications for the use of the AAQ-II in clinical and research contexts are discussed.