We have previously shown that exposure of P815 tumor cells to melphalan (l-phenylalanine mustard; l-PAM) leads to up-regulation of B7-1 surface expression, and this l-PAM-induced up-regulation requires de novo RNA synthesis and is associated with accumulation of B7-1 mRNA. Here we show that the effect of l-PAM on B7-1 surface expression can be mimicked by exposing P815 tumor cells to oxidative stress but not to heat shock. Moreover, the antioxidant N-acetyl-l-cysteine prevented the l-PAM-induced accumulation of B7-1 mRNA in P815 tumor cells, suggesting that reactive oxygen species are involved in the transcriptional regulation of l-PAM-induced B7-1 gene expression. Although AP-1 and NF-κB are regarded as redox-sensitive transcription factors and the promoter/enhancer region of the B7-1 gene contains an AP-1 and an NF-κB binding site, exposure of P815 tumor cells to l-PAM led to rapid and transient activation only of NF-κB, but not AP-1, that bound specifically to a probe containing the respective binding site in the murine or human B7-1 gene. Moreover, exposure of P815 tumor cells to a cell-permeable peptide that selectively inhibits NF-κB activation by blocking the activation of the IκB-kinase complex was found to inhibit the l-PAM-induced B7-1 mRNA accumulation, indicating that NF-κB activation is essential for the l-PAM-induced B7-1 gene expression. Taken together, these results indicate that l-PAM leads to activation of B7-1 gene expression by activating NF-κB via a pathway that involves reactive oxygen species.