An elevated blood level of homocysteine is associated with arteriosclerosis and thrombosis. The mechanisms by which homocysteine may promote vascular diseases have not been elucidated yet. In the present study, we have applied a modified nonradioactive differential display analysis to evaluate changes in gene expression induced by homocysteine treatment of cultured human umbilical vein endothelial cells (HUVEC). We identified six up-regulated and one down-regulated genes. One up-regulated gene was GRP78/BiP, a stress protein, suggesting that misfolded proteins would accumulate in the endoplasmic reticulum because of redox potential changes caused by homocysteine. Another up-regulated gene encoded a bifunctional enzyme with activities of methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase, which is involved in a homocysteine metabolism. A third up-regulated gene encoded activating transcription factor 4, and a fourth was a gene whose function is not identified yet. The remaining three were novel genes. We isolated a full-length cDNA of one of the up-regulated genes from a HUVEC library. It encoded a novel protein with 394 amino acids, which was termed reducing agents and tunicamycin-responsive protein (RTP). Northern blot analysis revealed that RTP gene expression was induced in HUVEC after 4 h incubation with homocysteine. RTP mRNA was also observed in unstimulated cells and induced by not only homocysteine but also 2-mercaptoethanol and tunicamycin. The mRNA was ubiquitously expressed in human tissues. These observations indicate that homocysteine can alter the expressivity of multiple genes, including a stress protein and several novel genes. These responses may contribute to atherogenesis.