Objective: In the kidney collecting duct, vasopressin signaling via the V2-receptor is responsible for regulation of several water and solute transport processes. The aim of this study was to identify signaling pathways involved in the short-term V2-receptor-mediated response in cultured collecting duct cells (mpkCCD). Methods: We used mass spectrometry-based quantitative phosphoproteomics combined with Stable Isotope Labeling by Amino acids in Cell culture (SILAC). Differentially labeled cell populations were treated with the V2-receptor specific agonist dDAVP (0.1nM) or vehicle for 30min. Results: 3900 phosphopeptides were identified and 2884 phosphopeptides were quantified. The majority (81.7%) of quantified phosphopeptides did not change in abundance in response to dDAVP. Analysis of the 273 phosphopeptides increased by dDAVP showed a predominance of so-called 'basophilic' motifs consistent with activation of kinases of the AGC family. Increases of phosphoryation of several known protein kinase A targets were found. In addition, increased phosphorylation of targets of the calmodulin-dependent kinase family was seen, including autophosphorylation of calmodulin-dependent kinase 2 at T286. Analysis of the 254 phosphopeptides decreased in abundance by dDAVP showed a predominance of so-called 'proline-directed' motifs, consistent with downregulation of mitogen-activated or cyclin-dependent kinases. dDAVP decreased phosphoryation of both JNK1/2 (T183/Y185) and ERK1/2 (T183/Y185; T203/Y205), consistent with a decrease in activation of these proline-directed kinases in response to dDAVP. Conclusion: The data support roles for multiple vasopressin V2-receptor-dependent signaling pathways in the vasopressin signaling network of collecting duct cells, involving several kinases not generally accepted to regulate collecting duct function. These kinases might also be responsible for long-term regulation of aquaporin-2 abundance.