Vasopressin regulates water excretion through effects on the renal collecting duct. Vasopressin signaling in the inner medullary collecting duct (IMCD) is mediated by V2-receptor occupation coupled to the generation of cyclic AMP (cAMP). Here, we employ a 'systems' approach to analysis of vasopressin signaling. The objective is to investigate roles of activation of the Akt and ERK1/2 MAP kinase pathways, as well as Ca²⁺ mobilization, in IMCD cells isolated from rat kidney. The V2-receptor selective vasopressin analog dDAVP increased the state of Akt activation (increased phosphorylation at T308 and S473) and decreased the state of ERK1/2 activation (decreased phosphorylation at T202 and Y204). Akt activation was blocked by an inhibitor of PI3K, LY294002. In microdissected IMCD segments, nonperiodic spike-like increases in intracellular Ca²⁺ (FLUO-4) were accelerated by vasopressin. Chelation of Ca²⁺ or calmodulin inhibition markedly decreased Akt phosphorylation. Decreased ERK1/2 phosphorylation was associated with a decrease in MEK1/2 phosphorylation and an increase in c-Raf phosphorylation at S259 (an inhibitory site). Based on the current findings integrated with previous findings in the IMCD, we now report a 33-node vasopressin-signaling network involved in vasopressin regulation of IMCD function.