Aquaporin-2 (AQP2) is a small, transmembrane protein, active in the principal cells of the renal collecting duct system. Phosphorylation and dephosphorylation of this protein regulate its redistribution in the cell, thereby influencing renal water reabsorption. The aims of the study were to identify and analyse the location of AQP2 phosphorylated at position 261, and to determine its potential role in renal water balance regulation, because data on AQP2 in cattle are scarce, and the mechanisms regulating its expression and cellular localization remain unclear. The analyses were carried out on kidneys collected from eight Polish Holstein-Friesian Black-and-White male calves, aged 5–7 months. It was found, using immunohistochemistry and commercially available phosphospecific antibodies, that in cattle AQP2 Ser-261 is located exclusively in the apical plasma membrane of the principal cells of the collecting duct. This atypical location of AQP2 Ser-261 allows to conclude that it may play a significant role in the process of renal water retention. The results of our previous studies on the regulation of renal total AQP2 excretion with urine in calves, as well as the bioinformatic analysis of available data presented in this study, seem to support this assumption. In addition, bioinformatic tools predicted mitogen activated protein kinases (MAPK) as a possible vasopressin-independent factor involved in AQP2 Ser-261 phosphorylation and accumulation in the apical plasma membrane.