Background Chronic fatigue syndrome (CFS) is a condition that may be associated with substantial disability. The Medical Outcomes Study Short-Form General Health Survey (SF-36) is an instrument that has been widely used in outpatient populations to determine functional status. Our objectives were to describe the usefulness of the SF-36 in CFS patients and to determine if subscale scores could distinguish patients with CFS from subjects with unexplained chronic fatigue (CF), major depression (MD), or acute infectious mononucleosis (AIM), and from healthy control subjects (HC). An additional goal was to ascertain if subscale scores correlated with the signs and symptoms of CFS or the presence of psychiatric disorders and fibromyalgia. Design Prospectively collected case series. setting Patients with CFS and CF were seen in a university-based referral clinic and had undergone a complete medical and psychiatric evaluation. Other study subjects were recruited from the community to participate in research studies. Participants The study included 185 patients with CFS, 246 with CF, 111 with AIM, and 25 with MD. There were 99 HC subjects. Measures The SF-36 and a structured psychiatric interview were used. The SF-36 contains 8 subscales: physical, emotional, social, and role functioning, body pain, mental health, vitality, and general health—and a structured psychiatric interview. Results Performance characteristics (internal reliability coefficients, convergent validity) of the SF-36 were excellent. A strikingly consistent pattern was found for the physical functioning, role functioning, social functioning, general health, and body pain subscales, with the lowest scores in CFS patients, intermediate scores in AIM patients, and the highest scores in the HC subjects. The CFS patients had significantly lower scores than patients with CF alone on the physical functioning ( P ≤0.01), role functioning ( P ≤0.01), and body pain ( P ≤0.001) subscales. The emotional functioning and mental health scores were worst among those with MD. The presence of fibromyalgia, being unemployed, and increasing fatigue severity all were associated with additional functional limitations across multiple functional domains, with increasing fatigue appearing to have the greatest effect. Conclusions The SF-36 is useful in assessing functional status in patients with fatiguing illnesses. Patients with CFS and CF have marked impairment of their functional status. The severity and pattern of impairment as documented by the SF-36 distinguishes patients with CFS and CF from those with MD and AIM, and from HC, but does not discriminate between CF and CFS.