The influences of dosing time and dosing schedule on the plasma alpha interferon (IFN-) concentration and the production of anti-IFN- neutralizing antibodies were investigated in ICR male mice adapted to cycles of 12 h of light and 12 h of dark. In mice pretreated with IFN- for 21 days, the plasma IFN- concentrations were significantly lower than those in control mice (P < 0.01). The clearance of IFN- and its volume of distribution obtained at steady state were significantly higher in the animals with IFN- pretreatment than in the mice without IFN- pretreatment. The area under the concentration-time curve and the mean residence time of IFN- were significantly smaller in IFN--pretreated animals than in control animals. The plasma IFN- levels (measured 2 h after dosing) were significantly lower in mice treated daily with IFN-, while the anti-IFN- neutralizing antibody levels (measured 24 h after dosing) were significantly increased on days 15 and 21 of treatment. Plasma IFN- levels were significantly decreased in association with the production of anti-IFN- neutralizing antibodies in mice treated with IFN- daily at either 0900 or 2100 h. By contrast, the plasma IFN- levels (measured 2 h after dosing) remained stable in mice treated with IFN- at 0900 h on alternate days, while they were significantly lower after 21 days of treatment in mice treated with IFN- at 2100 h on alternate days. These changes were associated with a significant increase in the levels of anti-IFN- neutralizing antibodies in the latter group. The present findings suggest that an appropriate dosing schedule and/or dosing time for IFN- may reduce the level of production of anti-IFN- neutralizing antibodies in experimental and clinical situations.