We previously reported that our cDNA microarray analysis of primary non-small cell lung carcinoma (NSCLC) could predict for patients at increased risk of cancer recurrence. From the result of this analysis, we selected 11 genes that were considered candidate prognostic marker genes and used the realtime reverse transcription polymerase chain reaction (RT-PCR) to investigate their expression in the same set of NSCLC cases used in the microarray study. Cluster analysis of the realtime RT-PCR data separated these patients into two groups with significantly different disease-free survivals (log-rank test, P < 0.017). In contrast, cluster analysis failed to confirm the prognostic significance of the realtime RT-PCR results for these 11 genes in a validation series of 92 NSCLC cases. In univariate analysis, hypoxia inducible factor 1alpha, Rho-GDP dissociation inhibitor (GDI) alpha (RhoGDI) and Citron/rho-interacting serine-threonine kinase 21 (Citron K21) were significant prognostic factors for disease-free survival in the entire cohort of 130 NSCLC patients, but none were significant in multivariate analysis. The results demonstrate that the prognostic significance of microarray (SAM) results can be partially validated using realtime RT-PCR, but secondary validation using larger and independent series of tumors is necessary to identify true prognostic marker genes.