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Introduction: In sera of pituitary disease patients and other autoimmune endocrine disease are detectable pituitary autoantibodies. Until now characterization of pituitary antigen is still unknown. The aim of our study was isolation and characterization of pituitary autoantigen by affinity chromatography. For isolation we have used microsomal fraction of human pituitary., Material and Methods: For immunoglobulins isolation have been used sera of pituitary disease, Addison disease and Graves-Basedow disease patients with detectable pituitary autoantibodies. For pituitary antigen isolation have been used microsomal fraction of human pituitary obtained by ultracentrifugation and solubilisation. Immunoglobulins isolation was performed on Sepharose 4B-Protein A. Immunosorbent was performed on CNBr-activated Cl-4B Sepharose. Desorbtion was conducting by 0.2 mol/L glicine and 1 mol/L, 3 mol/L guanidine. The estimation of isolated proteins was performed by immunoblotting., Results: Isolation of immunoglobulins from patients sera was done 12 times receiving from 8.5 up to 13.5 mg of IgG from 1-1.5 ml of sera. In desorbtion we have received from 0.026 up to 0.150 mg of antigen proteins. For molecular weight estimation isolated proteins have been labeled by (125)I and run on SDS/PAGE with autoradiography. Autoradiography shown us two lines with 67 kDa and 55 kDa and low weight protein line., Conclusions: Isolation of pituitary autoantigen by affinity chromatography shown two different antigen proteins with 67 kDa and 55 kDa.