Studies by many investigators have demonstrated that the immune system is subject to regular circadian fluctuation. Some rhythms that have been reported include circadian changes in components of the immune system, e.g., lymphocytes, and circadian variation in primary and secondary immune responsiveness. The observation that many of these rhythms are inversely correlated to the glucocorticoid rhythm has led to the suggestion that fluctuations in the immune system may be a result of the glucocorticoid circadian rhythm. This study was designed to see if thymosin-alpha 1 (Tsn-alpha 1), a 28-amino acid polypeptide isolated from bovine thymus that has been reported to influence thymocyte differentiation, might follow a circadian rhythm, and thus play a role in the periodicity of the immune system. In these experiments, groups of 10 C57BL/6 or Swiss Webster mice were sacrificed at 4- or 6-hr intervals over a 24-hr period. Serum Tsn-alpha 1 and corticosterone levels were determined by radioimmunoassay. Results from the first experiment showed that Tsn-alpha 1 undergoes a circadian rhythm (p less than 0.001) with an acrophase (time of peak levels) 1.5 hr after the onset of light, and an amplitude (amount of maximum variation from the 24-hr mean) of 0.493 ng/ml Tsn-alpha 1-like immunoreactivity. These results were confirmed in an experiment in which the animals were placed on a reversed light cycle. In a separate experiment, the Tsn-alpha 1 circadian rhythm persisted in mice thymectomized 6 mo. earlier. In this latter experiment, a significant increase in the amplitude of the corticosterone rhythm in the thymectomized relative to sham-operated controls was also observed. Although these experiments do not imply casuality, it is interesting that the time of peak Tsn-alpha 1 levels can be correlated with the time of optimal immune function.