To establish a convenient, cost-effective, and reasonably reliable method for monitoring multiple gene expression using customized membrane-based macroarray, we constructed a cDNA macroarray with multiple probes for 13 human vascular endothelial genes and assessed the accuracy of the macroarray measurements. For each gene, two cDNA probes (450-550 bp) were designed from different regions (coding region and 3'-untranslated region [3'-UTR], respectively) on the basis of simple criteria concerning length and sequence specificity and spotted on the macroarray. In addition, unmodified oligonucleotide probes (80 mer) targeted to a unique sequence from the coding region of each gene were spotted on the same macroarray. Using this macroarray, shear stress-induced mRNA expression changes were analyzed in human coronary artery endothelial cells. Comparison of the expression ratios obtained with those measured using quantitative realtime polymerase chain reaction (PCR) as a reference method revealed that cDNA probes designed from a sequence within the coding region provided a highly accurate expression profile, whereas results obtained from oligonucleotide probes showed no correlation with real-time PCR data, which might be caused by inadequate immobilization of oligonucletotide probes on the nylon membrane. In addition, we observed that cDNA probes targeting different regions of a gene yielded different signal intensities. Most cDNA probes designed from a sequence within the coding region showed detectable signals, whereas few cDNA probes designed from 3'-UTR did.