A radioimmunoassay (RIA) for human thyroxine-binding prealbumin (PA) is described. It employs highly purified PA, anti-human PA serum at 1:30,000 final dilution, normal bovine serum as a carrier, and polyethyleneglycol to precipitate the immune complexes. This assay is extremely sensitive (limit of detection less than 0.2 micrograms per dL or less than 3.6 X 10(-15) moles per tube), accurate (recovery = 98.7 +/- 9 percent, mean +/- S.D.) and reproducible (intra- and inter-assay coefficients of variation = 3.6 to 6.3 percent and 7.2 to 9.5 percent, respectively). There was a highly significant correlation when the RIA was compared with radical immunodiffusion or with PA maximal binding capacity for thyroxine (r = 0.944 and r = 0.724, respectively, p less than 0.001). Concentration of PA in sera from normal subjects (age range = 20 to 88 years) averaged 27.7 +/- 0.5 mg per dL (mean +/- S.E.M.), with significantly higher values in males than in females in all age groups with the exception of the older subjects (20 to 50 years: males = 26.5 to 37 mg per dL; females = 23.1 to 33.8 mg per dL). Levels of PA progressively declined after the fifth decade of life. Pregnancy, hyperthyroidism, chronic liver diseases, cystic fibrosis, cancer and other non-thyroidal illnesses were associated with decreased levels of serum PA. Untreated hypothyroidism and chronic renal diseases showed widely scattered values of PA. Inherited thyroxine-binding globulin (TBG) abnormalities and bisalbuminemia had no apparent effect on concentrations of serum PA.