We measured the serum concentration and relative distribution of various free fatty acids (FFA) by gas/liquid chromatography in normal subjects and nonthyroid illness (NTI) patients with or without detectable serum thyroid-hormone binding inhibitor (THBI). The mean serum concentration of total FFA was 0.72 +/- 0.08 (SE) mM in eight normal subjects; it was similar (0.63 +/- 0.12) in eight THBI-negative NTI patients, but was significantly higher (3.1 +/- 1.0; P less than 0.05) in eight THBI-positive NTI patients (THBI index, 3.9 +/- 0.3 vs. 1.0 +/- 0.05 in normal subjects). Relative distribution of FFA in THBI-negative patients did not differ significantly from that in normal subjects. In THBI-positive patients, however, serum concentrations of palmitic, palmitoleic, stearic, and oleic acids were significantly above normal. Among fatty acids with appreciable THBI activity, oleic acid was most abundant in THBI-positive patients; its concentration of 1.3 +/- 0.32 mM in patients was about 6-fold higher than the corresponding normal value (0.21 +/- 0.02; P less than 0.005). The serum concentration of THBI correlated significantly with levels of both total FFA (r = 0.69; P less than 0.005) and oleic acid (r = 0.80; P less than 0.001). Addition of 0.33 mM oleic acid to THBI-negative NTI serum or 0.66 mM oleic acid to normal serum increased THBI activity to a level greater than 2 SD above the normal mean, i.e. 1.6. The various data suggest that 1) circulating FFA contribute importantly to THBI activity in sera of NTI patients; 2) oleic acid contributes more to THBI activity of NTI sera than do other FFA.