We are developing sets of mouse cDNAs with low redundancy for use in microarray analysis. The sets are based on sequence information from GenBank and the Washington University/Merck EST sequencing project. Related sequences from the non-redundant GenBank database were compared with mouse EST clones from the IMAGE consortium. A single EST for a given cluster was selected, colony purified and sequence validated. To evaluate this set of cDNAs for its use in differential gene expression studies, we generated cDNA microarrays. Those microarray slides were used in hybridization experiments using samples of the melanocyte-specific lineage of the neural crest as a model system. Gene expression was compared between RNA samples obtained from mouse melanoma tissue samples of a transgenic hepatocyte growth factor/scatter factor melanoma mouse model and a MEF-derived mouse fibroblast cell line (NIH3T3). Applying a confidence level of 99%, 8.5% of the arrayed genes were found to be differentially expressed. The melanocyte lineage marker genes Dct, Pmel17 and Sox10 are overexpressed in the melanoma samples with a ratio of 7.6, 6.9 and 7.8, respectively. A low expression ratio of 0.6 is seen for the fibroblast marker gene fibronectin. Genes encoding ubiquitin and the heat shock protein Hsc73 display no significant expression changes between the two differential samples.