Detection systems for chicken anemia virus (CAV) antigen in paraffin sections were evaluated. Mouse monoclonal antibodies to CAV used in conjunction with an avidin-biotin-peroxidase-complex detection system gave best results. Immunoreactivity of CAV was markedly affected by fixation. Fixation in neutral buffered formalin for 6 hours gave best results. Use of decalcifying fluid containing formic acid eliminated immunoreactivity of CAV, whereas use of an EDTA solution did not. In a sequential study, CAV antigen and lesions were first detected in bone marrow, thymus, and spleen at days 3-4 postinoculation (PI). Subsequently, antigen and/or cells containing nuclear inclusions were found in many tissues, but usually within lymphoid tissue therein. Thymus, spleen, bone marrow, proventriculus, and ascending duodenum contained most antigen. No antigen was detected after 26 days PI. The results indicated that CAV replicates in thymic lymphoblasts, intra- and extra-sinusoidal hemocytoblasts, and reticular cells, with consequent lymphocytic depletion of the thymic cortex and hypoplasia of the bone marrow, and that CAV antigen is widely distributed in the body.