NY-ESO-1 is a cancer/testis (CT) antigen expressed in normal adult tissues solely in the testicular germ cells of normal adults and in various cancers. It induces specific humoral and cellular immunity in patients with NY-ESO-1-expressing cancer. We compared the expression of NY-ESO-1 mRNA in hepatocellular carcinoma (HCC) patients by using various primers and DNA polymerases to optimize RT-PCR conditions and to evaluate the correlations among the expression levels of NY-ESO-1, LAGE-1 and SSX-1 and clinical parameters. We determined differences in the abilities of the various primers and DNA polymerases to amplify the NY-ESO-1 gene at different exons. Primers designated as P3 detected targeted sequences better than primers P1 and P2; AmpliTaq Gold DNA polymerase was more effective than Platinum pfx DNA polymerase and Taq DNA polymerase. NY-ESO-1, LAGE-1 and SSX-1 mRNAs were detected in 29.7, 45.3 and 37.5%, respectively, of the 64 HCC specimens. No CT antigen mRNAs were detected in the 64-paired adjacent non-cancerous tissues. The frequency for the co-expression of one, two or three antigens of NY-ESO-1, LAGE-1 and SSX-1 was 57.8, 35.9 and 18.8%, respectively. We also analyzed the relationships among the CT antigen expression levels and several clinical parameters. There were no significant differences between CT antigen expression levels and clinical parameters, except the correlations between the expression of SSX-1 and the age of the patients.