Peptides have been found in a variety of tissues including brain. To purify the peptide angiotensin II, a three-step method for the isolation and purification has been developed using extraction, affinity chromatography, and high-pressure liquid chromatography. Angiotensin II antiserum purified by affinity chromatography was covalently coupled to Affi-gel 10 (Affi-gel IO-AB). The efficiency and usefulness of this column for the purification of angiotensin II from biological sources were tested with 1251-and 'H-labeled (Ile5)-angiotensin II added to rat brains prior to extraction. After extraction, the recoveries for both peptides were 74 and 752, respectively. Recovery after the purification on Al&gel IO-AB was 84 and 82%. Thirty-two percent of the radioactivity was not retained and 50% of the radioactivity could be eluted with 0.1 M Na citrate buffer containing 1 M NaCl using a stepwise pH gradient. Characterization by HPLC of the unretained radioactivity from the Affi-gel lo-AB column showed one peak for ['*'I]angiotensin II, coeluting with the ['251]angiotensin II standard and two minor peaks. Only 30% of unretained ["Hlangiotensin II could be identified as intact [3H]angiotensin II on HPLC. Both ['251]angiotensin II and [3H]angiotensin II elutable at pH 5.0 and 4.0 on Affi-gel IO-AB could be demonstrated as highly purified ['*'I]angiotensin II and [3H]angiotensin II on HPLC with a purity of more than 90%. On HPLC, the recovery was 8 1% for ['*'I]angiotensin II and 99% for [3H]angiotensin II. The recovery for the entire three-step procedure was about 60%. The loading capacity of the Affi-gel lo-AB column for (Ile5)-angiotensin II was 550 ng. With the method described, angiotensin II could be purified very specifically and in a high purity. The method may also be used for the rapid purification of other neuropeptides. 0 1986 Academic press. IX.