beta-Arrestins are multifunctional adaptor proteins that regulate seven transmembrane-spanning receptor (7TMR) desensitization and internalization and also initiate alternative signaling pathways. Studies have shown that beta-arrestins undergo a conformational change upon interaction with agonist-occupied, phosphorylated 7TMRs. Although conformational changes have been reported for visual arrestin and beta-arrestin2, these studies are not representative of conformational changes in beta-arrestin1. Accordingly, in this study, we determine conformational changes in beta-arrestin1 using limited tryptic proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis in the presence of a phosphopeptide derived from the C terminus of the V(2) vasopressin receptor (V(2)Rpp) or the corresponding unphosphorylated peptide (V(2)Rnp). V(2)Rpp binds specifically to beta-arrestin1 causing significant conformational changes, whereas V(2)Rnp does not alter the conformation of beta-arrestin1. Upon V(2)Rpp binding, we show that the previously shielded Arg(393) becomes accessible, which indicates release of the C terminus. Moreover, we show that Arg(285) becomes more accessible, and this residue is located in a region of beta-arrestin1 responsible for stabilization of its polar core. These two findings demonstrate "activation" of beta-arrestin1, and we also show a functional consequence of the release of the C terminus of beta-arrestin1 by enhanced clathrin binding. In addition, we show marked protection of the N-domain of beta-arrestin1 in the presence of V(2)Rpp, which is consistent with previous studies suggesting the N-domain is responsible for recognizing phosphates in 7TMRs. A striking difference in conformational changes is observed in beta-arrestin1 when compared with beta-arrestin2, namely the flexibility of the interdomain hinge region. This study represents the first direct evidence that the "receptor-bound" conformations of beta-arrestins1 and 2 are different.