Vasopressin-regulated urea transport in the renal inner medullary collecting duct (IMCD) is mediated by two urea channel proteins, UT-A1 and UT-A3, derived from the same gene (Slc14a2) by alternative splicing. The NH(2)-terminal 459 amino acids are the same in both proteins. To study UT-A1/3 phosphorylation, we made phospho-specific antibodies to UT-A sequences targeting phospho-serines at positions 84 and 486, sites identified previously by protein mass spectrometry. Both antibodies proved specific, recognizing only the phosphorylated forms of UT-A1 and -A3. Immunoblotting of rat IMCD suspensions or whole inner medullas showed that the V2R-selective vasopressin analog 1-deamino-8-d-arginine vasopressin (dDAVP) increases phosphorylation at Ser84 (in UT-A1 and UT-A3) and Ser486 (in UT-A1) by about eightfold. Time course studies in rat IMCD suspensions showed maximum phosphorylation within 1 min of dDAVP exposure, consistent with the time course of vasopressin-stimulated phosphorylation of the vasopressin-sensitive water channel aquaporin-2 at Ser256. Confocal immunofluorescence in Brattleboro rat medullary tissue showed labeling limited to the IMCD, which increased markedly in response to dDAVP. Immuno-electron microscopy studies showed that both phosphorylated forms were present mainly in intracellular compartments in the presence of vasopressin. These studies demonstrate regulated phosphorylation of both UT-A1 and UT-A3 in response to vasopressin in a manner consistent with coordinate regulation of UT-A and aquaporin-2 in the renal IMCD. The findings add to prior evidence for vasopressin-induced phosphorylation of UT-A1, providing evidence that UT-A3 may be regulated by phosphorylation as well.