Previous studies from this laboratory have demonstrated a statistically significant dysregulation in several key components of the 2',5'-oligoadenylate (2-5A) synthetase/RNase L and PKR antiviral pathways in chronic fatigue syndrome (CFS) (Suhadolnik et al. Clin Infect Dis 18, S96-104, 1994; Suhadolnik et al. In Vivo 8, 599-604, 1994). Two methodologies have been developed to further examine the upregulated RNase L activity in CFS. First, photoaffinity labeling of extracts of peripheral blood mononuclear cells (PBMC) with the azido 2-5A photoaffinity probe, [32P]pApAp(8-azidoA), followed by immunoprecipitation with a polyclonal antibody against recombinant, human 80-kDa RNase L and analysis under denaturing conditions. A subset of individuals with CFS was identified with only one 2-5A binding protein at 37 kDa, whereas in extracts of PBMC from a second subset of CFS PBMC and from healthy controls, photolabeled/immunoreactive 2-5A binding proteins were detected at 80, 42, and 37 kDa. Second, analytic gel permeation HPLC was completed under native conditions. Extracts of healthy control PBMC revealed 2-5A binding and 2-5A-dependent RNase L enzyme activity at 80 and 42 kDa as determined by hydrolysis of poly(U)-3'-[32P]pCp. A subset of CFS PBMC contained 2-5A binding proteins with 2-5A-dependent RNase L enzyme activity at 80, 42, and 30 kDa. However, a second subset of CFS PBMC contained 2-5A binding and 2-5A-dependent RNase L enzyme activity only at 30 kDa. Evidence is provided indicating that the RNase L enzyme dysfunction in CFS is more complex than previously reported.