Antigen-binding peripheral blood leukocytes (PBL) from normal and bursectomized agammaglobulinemic chickens were labeled by incubation in vitro with radioiodinated antigen at 4 degrees C in the presence of sodium azide. [125I]TGAL-binding cells could be detected by autoradiography of PBL from normal, unimmunized chickens at a frequency of 1 to 4 labeled cells per 10(4) leukocytes. No [125I]TGAL-binding cells were found in PBL from bursectomized chickens, even after incubation with 25 mug/ml of labeled antigen followed by prolonged autoradiographic exposure. The binding to normal PBL was specific as judged by inhibition with unlabeled TGAL but not with unlabeled TIGAL. The binding was, furthermore, inhibited by preincubation with rabbit anti-chicken L chain antibody but unaffected by normal rabbit IgG. [125I]TIGAL was, in contrast, found to bind to PBL from both normal and bursectomized chickens at a frequency of 6 to 80 labeled cells per 10(4) leukocytes. The labeling was specific, since it was inhibited by cold TIGAL but not by cold TGAL. The binding of [125I]TIGAL to PBL from bursectomized chickens showed from none to slight inhibition on preincubation of the cells with anti-L chain antibody, whereas preincubation with normal rabbit IgG resulted in almost complete inhibition. To our knowledge this is the first demonstration of antigen binding to PBL from agammaglobulinemic chickens.