The expression profiles of thousands of genes can be analyzed simultaneously by hybridizing labeled complementary DNA targets to microarrays. Two-color hybridization allows comparison of two samples labeled with different fluorescent dyes on the same microarray. Normalized signals are expressed as the ratio of signals from Cy5 experimental and Cy3 reference probes. Use of a common reference sample across multiple experiments provides reliable data comparison. A reference sample should provide a hybridization signal at as many probe elements as possible. We chose 21 human cell lines, representing different tissues, to approximate the expression profile of all human genes. The expression profile of each individual cell line and that of a pool of RNAs from 21 cell lines were compared. Hybridization was performed using 24K probe element human microarrays (Stanford University) and GeneConnection Discovery microarrays (Stratagene). Signal distribution on these microarrays demonstrates that reference cDNA hybridizes to a larger number of tethered probes than cDNA from any single cell line. Ten different cell lines were chosen for Universal Human Reference RNA, providing optimal coverage on human microarrays; these can be used as a reference sample for any microarray experiment.