The biological activities of molecules secreted into the serum of mice chronically irradiated with γ rays at low or medium dose rate (L/MDR) have not been well studied. In this work, the bioactive molecules found in the serum of chronically irradiated mice (dose rate: 0.0181 Gy/h) were characterized by a cell-based assay (CBA) using microarrays. This technique can predict changes in cytokine levels in serum by measuring gene expression profiles and analyzing molecular signaling pathways. Gene expression in cultured mouse embryo fibroblasts (MEFs) 1 day after addition of serum from nonirradiated or irradiated mice had different profiles. A high level of expression of lipocalin2 (Lcn2) was induced in MEFs upon addition of serum from MDR irradiated mice, and Lcn2 was used as a marker for identifying secreted molecules in serum. Based on microarray analysis of molecular pathways, we predicted that the enhanced gene expression of Lcn2 in MEFs might be caused by interleukin-1 (IL-1) in the serum of the irradiated mice, and that an IL-1α antibody could completely neutralize the enhanced gene expression of Lcn2 in MEFs. The increase in IL-1α levels in the serum from the irradiated mice was confirmed by ELISA experiments. However, an increase in IL-1β could not be detected. These results indicated that IL-1α was released into the serum of mice chronically exposed to a high dose of γ-ray radiation at MDR. We therefore believe that the CBA method using microarrays will be applicable for the screening of bioactive molecules in serum, which will be useful for detecting various diseases and metabolic changes.