We have used both the UniGene RIKEN epididymal EST library and the Affymetrix microarray profiling for identifying novel epididymal genes in mouse. The use of ESTs is a complementary approach to Affymetrix arrays for identifying novel epididymal genes, while only 32% and 28% of ESTs of unknown genes were present in the U74v.2Set and MG 430 2.0 version Affymetrix arrays, respectively. As expected, the probe set for a notably larger proportion of known genes was present in the Affymetrix arrays, and the coverage was greatly improved by the newer array version. Furthermore, many genes with more than five ESTs in the UniGene library showed variable levels of expression in both versions of the Affymetrix arrays. However, both the Affymetrix and EST data correlated well with that obtained by quantitative RT-PCR, and thus, we conclude that the findings of high EST number but only limited expression in the arrays could be considered as false negatives in the Affymetrix arrays.