We have performed genome-wide expression profiling of endocrine regulation of genes expressed in the mouse initial segment (IS) and distal caput of the epididymis by using Affymetrix microarrays. The data revealed that of the 15 020 genes expressed in the epididymis, 35% were enriched in one of the two regions studied, indicating that differential functions can be attributed to the IS and the more distal caput regions. The data, furthermore, showed that 27% of the genes expressed in the IS and/or distal caput epididymidis are under the regulation of testicular factors present in the duct fluid, while bloodborne androgens can regulate for 14% of them. This is in line with the high testis dependency of epididymal physiology. We then focused on genes with moderate or strong expression, showing strict segment enrichment and strong dependency on testicular factors. Analyses of the 59 genes, including upregulated and downregulated genes, fulfilling the criteria indicated that the expression of 18 (17 downregulated genes; 1 upregulated gene) of 19 gonadectomy-responsive genes enriched in the IS was not maintained by the androgen treatment, whereas the expression of all six downregulated genes enriched in the distal caput and the majority of those with no strict segment enrichment of expression (28 of 34; consisting of 23 downregulated and 5 upregulated genes) were maintained by androgens. Hence, it is evident that testicular factors other than androgens are important for the expression of IS-enriched genes, whereas the expression of distal caput-enriched genes is typically regulated by androgens. Identical data were obtained by independent clustering analyses performed for the expression data of 3626 epididymal genes. Several novel genes with putative involvement in epididymal sperm maturation, such as a disintegrin and metallopeptidase domain 28 (Adam28) and a solute carrier organic anion transporter family, member 4C1 (Slco4c1), were identified, indicating that this approach is successful for identifying novel epididymal genes.