The region of the arginine-vasopressin (AVP) molecule critical for binding to the effective antibodies in a RIA has been localized to the vicinity of the Phe3 position by using the cross-reaction in the assay between AVP and a number of its structural analogs. Binding seems to be almost independent of any direct contributions from components of the tripeptide tail of AVP. Using this RIA it was found that disequilibrium conditions of incubation produce a 5-fold increase in assay sensitivity over equilibrium conditions. Amino acid analysis revealed that three synthetic peptide preparations used as reference standards comprised only 70-80% of their weight as peptide and this finding points up the need to correct such reference standards for their peptide content. The ratio of rat vasopressor activity to RIA activity of these three preparations as well as of a natural AVP preparation, however, approximated unity. Results obtained comparing measurements of AVP in rat neural lobes by RIA and rat vasopressor assay show a correlation between RIA and bioassay of 0.9406, a slope of 1.086, and an intercept of 20 mU, suggesting good agreement for AVP determined by these two assay systems.