We investigated NY-ESO-1 and LAGE-1a mRNA expression in normal tissues and various types of cancer by quantitative real-time RT-PCR. In addition to their high expression in the testis, we observed a low expression of NY-ESO-1 mRNA in the placenta, pancreas and liver, and no expression in 12 other normal tissues. We also observed a low expression of LAGE-1a mRNA in the placenta and ovary, and marginal expression in 13 other normal tissues. In contrast to the previous finding that NY-ESO-1 and LAGE-1a mRNAs were mostly co-expressed in solid tumors, we found an independent expression of NY-ESO-1 and LAGE-1a mRNAs. NY-ESO-1 mRNA expression was mostly associated with LAGE-1a mRNA expression in esophageal and liver cancers, but not in prostate cancer. Immunohistochemistry (IHC) using NY-ESO-1-specific ES121 mAb showed that NY-ESO-1 protein was detected in 6 of 9 and 3 of 10 NY-ESO-1 mRNA-positive specimens from esophageal and liver cancers, respectively. NY-ESO-1 protein expression was correlated with the copy numbers of NY-ESO-1 mRNA. IHC was also performed using ES121 mAb and B9.8 mAb recognizing both NY-ESO-1 and LAGE-1a in 4 esophageal and 6 liver cancer specimens preferentially expressing LAGE-1a mRNA. B9.8-specific staining was observed weakly and focally in one liver cancer specimen expressing >10(5) copies of LAGE-1a mRNA.