OBJECTIVE Recent work both from our laboratory and in marsupial models of testicular descent suggests a strong connection between the mammary line and gubernacular migration. This study investigated the relationship between the mammary fat pad (MFP) that underlies the mammary line and the developing gubernaculum by fluorescent immunohistochemistry. METHODS Rats at E17 and E19 were fixed and processed for immunohistochemistry. Sagittal sections of male fetuses were stained with antibodies against androgen receptor (AR), prolyl-4 hydroxylase β, Desmin, activated Notch-1, Jagged-1, and Ki-67. These were analyzed by fluorescent confocal microscopy. RESULTS At E17 and E19, the MFP anlage forms a continuous distribution of fibroblasts passing immediately adjacent the gubernaculum to the future scrotum. Within this exists a distinct subpopulation of fibroblasts expressing AR distributed over the path of inguinoscrotal descent of the gubernaculum. Proliferation and Notch-1 signaling were similar throughout the MFP with differential Notch-1 signaling in the E19 gubernaculum. CONCLUSIONS This investigation has identified the presence of a distinct AR-expressing subpopulation of MFP fibroblasts over the path of inguinoscrotal descent during the key androgenic programming window of this phase. This unique developmental pattern is consistent with a prime role for the MFP in testicular descent.