A radioimmunoassay for circulating alpha-interferon (IFN alpha) has been developed using lymphoblastoid IFN alpha. The assay was specific for IFN alpha, and did not cross-react with IFN beta, IFN gamma, or ACTH, while it was specifically inhibited by recombinant IFN alpha. The radioimmunoassay (y) correlated linearly with the virus inhibition assay (x), with a regression line of y on x of y = 0.659x + 245 (u) (P less than 0.01). alpha-Interferon-like substance (IFN alpha-LS) was extracted and concentrated from plasma either by silicic acid or by antibody immunoadsorption. Serial dilutions of plasma and extracted samples of plasma showed dilution curves identical to those of standard IFN alpha, suggesting the presence of endogenous IFN alpha in human plasma. The circulating IFN alpha-LS of healthy individuals aged 20 to 45 was 0.207 +/- 0.055 ng/ml in males (n = 48) and 0.172 +/- 0.076 ng/ml in females (n = 34). Gel filtration studies on a Sephadex G-75 column suggested that circulating IFN alpha-LS exists in a fragmented form, inactive in virus inhibition assays, in the plasma of healthy individuals. The finding may help explain why biological IFN alpha is often undetectable in the plasma of healthy donors, yet is detectable by radioimmunoassay. Circulating IFN alpha-LS in the plasma of healthy individuals declined gradually with age. IFN alpha-LS was significantly decreased in the plasma of rheumatoid arthritis patients, when compared with the value found in the age and sex-matched healthy controls and in osteoarthritis patients (P less than 0.0001). The decrease was related neither to treatment nor to disease activity. IFN alpha-LS was, however, not decreased in the plasma of vasculitis patients. Decreased IFN alpha-LS in rheumatoid arthritis may be important from pathogenetic and therapeutic standpoints.