Methodological issues in the use of biologic markers in research on the chronic fatigue syndrome (CFS) were discussed. The need for control and limitation of measurement errors, and the need for reliability of tests were addressed. The validity of a marker was critical in that it clearly represented the phenomenon being measured. The use of biologic markers in research required their validation as dependent or independent variables. The no effect threshold and the positive predictive value (PPV) of a marker were discussed. In population studies of CFS in which prevalence was low, the specificity of a marker weighed heavily in determining the PPV. Confounding factors, legal and ethical implications, base levels, and background levels were important considerations before use of markers. A checklist was provided for evaluating a biologic marker which included the availability of evidence for the validity of the marker, conditions necessary, multiple markers, and impact on the subject. The author cautions that biologic markers should only be considered tools to be added to the battery of those already existing, and that they should not be thought of as conclusive by themselves.