The interferon-induced intracellular enzyme, 2',5'-oligoadenylate (2,5A) synthetase, was measured in extracts of Ficoll-purified human peripheral mononuclear cells from 28 normal healthy individuals and 14 patients receiving injections of interferon-alpha. Basal and stimulated levels could be measured reproducibly in 2 X 10(6) cells. In both groups, mononuclear cell levels of 2,5A synthetase varied widely, but measurements were reproducible in each individual. In most instances, increases in enzyme activity were detected within 8 h of interferon-alpha administration. Elevated levels persisted for at least 24 h and were maintained with daily treatment. In two of the 14 patients, the enzyme level failed to respond to multiple interferon injections. Interestingly, these two patients had pretreatment enzyme levels that were markedly elevated relative to those of a healthy population. Measurement of 2,5A synthetase levels is a valuable addition to clinical interferon studies, since the results may help to resolve questions of the optimal dose, route, and schedule of interferon administration.