We have previously demonstrated irradiation-induced up-regulation of CD80 expression in A20-HL B lymphoma cells by inducing expression of tumour necrosis factor-α (TNF-α) and CD154. In the present study, we investigated whether irradiation also up-regulates CD80 expression in mouse spleen B cells. Because freshly prepared spleen B cells are highly sensitive to irradiation, we employed spleen B cells stimulated with lipopolysaccharide (LPS-B cells). X-irradiation (8 Gy) followed by incubation (9–12 hr) highly and selectively up-regulated CD80 expression in LPS-B cells, whereas the same treatment slightly increased expression of CD54 and did not affect expression of CD86, major histocompatibility complex class II, CD11a or surface immunoglobulin M. The irradiation-induced up-regulation of CD80 expression resulted in enhanced APC function of LPS-B cells. Up-regulation of CD80 expression on LPS-B cells was accompanied by an increase in CD80 mRNA accumulation and nuclear factor (NF)-κB activation. Activation of NF-κB was shown to be critical for up-regulation of CD80 expression as pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, severely decreased the observed up-regulation. X-irradiation of LPS-B cells induced expression of TNF-α but not CD154. However, anti-TNF-α monoclonal antibody (mAb) with anti-CD154 mAb did not inhibit X-irradiation-induced up-regulation of CD80 expression in LPS-B cells, whereas these mAbs almost completely inhibited this up-regulation in A20-HL cells and bone marrow-derived dendritic cells (DCs). In contrast, a thiol antioxidant, N-acetyl-l-cysteine, completely blocked X-irradiation-induced up-regulation of CD80 expression in LPS-B cells, but not in A20-HL cells or in DCs. Based on these findings, we concluded that X-irradiation up-regulates CD80 expression not only in A20-HL cells and DCs but also in LPS-B cells, and that this up-regulation in LPS-B cells via NF-κB activation is dependent on the generation of reactive oxygen species, while that in A20-HL cells and DCs is not.