Androgens are the primary regulators of epididymal structure and functions. Principal cells are the major cell type of the tissue and are particularly sensitive to androgen removal. To distinguish the direct effects of androgens on this cell type, DNA microarrays were used to identify androgen-regulated genes in the mouse proximal caput epididymidis (PC-1) cell line. PC-1 cells display tissue- and caput- specific gene expression. We examined changes in gene expression occurring 2, 4, and 6 days following androgen deprivation and 2 days following androgen supplementation after being deprived of androgen for 2 or 4 days. Changes in transcript levels were investigated for mediators of androgen action; selected genes were analyzed by real-time RT-PCR, and changes at the protein levels were examined. Four distinct patterns of gene expression were activated following androgen withdrawal; the vast majority of genes displayed an early or late transient increase in expression levels. A differential ability of rescue was seen among androgen-regulated genes depending on time of androgen supplementation. Many of the genes that were rescued at 4 days were functionally linked by direct interactions and converged on IGF1. The ability for rescue after 4 days of androgen deprivation was severely compromised in many genes belonging to specific functional gene families (cell adhesion, cell growth, apoptosis, and cell cycle) and may be mediated in part by changes in AR coregulator expression. These results provide novel insights into the mechanisms of androgen regulation in epididymal principal cells.