Active proton secretion and bicarbonate reabsorption by epithelial cells of the mammalian excurrent duct system maintains an acidic luminal pH that is involved in creating a suitable environment for sperm maturation and storage. Both an apical Na/H exchanger and an apical H+ ATPase have been implicated in luminal acidification. The H+ ATPase is located in apical and/or narrow cells in the caput epididymidis, and clear cells in the corpus and cauda epididymidis. As a step toward understanding the acute and chronic regulation of luminal acidification in excurrent ducts, we have followed the appearance of H+ ATPase-rich cells in rat epididymis during postnatal development, using antibodies to subunits of the H+ ATPase. In addition, we performed double staining with antibodies against carbonic anhydrase type II (CAII). H+ ATPase-rich cells were already detectable 2 weeks after birth in all regions of the epididymis, and reached maximum numbers after 3-4 weeks. CAII-rich cells followed a similar developmental pattern. In adult rats, the number of H+ ATPase/CAII-positive cells in the cauda was on average more than double the number in the caput epididymidis, although considerable intertubule variability was seen in both regions. Double immunostaining showed that CAII and H+ ATPase were colocalized in the same cells in the caput and cauda, but H+ ATPase-rich cells in the corpus contained low levels of CAII. These results demonstrate that differentiated subpopulations of proton-secreting epithelial cells appear early during epididymal development, and that the induction of H+ ATPase in these cells occurs prior to sexual maturation.