The head-associated lymphoid tissues (HALT) of the chicken are composed of the Harderian gland and the conjunctiva-associated lymphoid tissue (CALT). Since these lymphoid tissues are potential immunological sites to be exposed to respiratory disease agents and intraocular and aerosol vaccines, lymphocytes of the HALT were studied. Methods employing in vitro and in situ techniques were used to examine the lymphocytes of the HALT. An in vitro lymphoproliferative assay was optimized to measure mitogenic responses of lymphocytes isolated from the spleen, peripheral blood, and Harderian gland. Harderian gland lymphocytes responded to T and B cell mitogens as did the lymphocytes from the spleen and blood. Mitogenic responses of the lymphocytes isolated from CALT could not be measured by the described methodology. An in situ immunocytochemical technique was used to identify lymphocyte subpopulations of the HALT as the chicken aged. The concentration of T cells, particularly T cells expressing pan T cell and T helper cell epitopes, within the Harderian gland increased with age; while the B lymphocyte population remained unchanged. Aggregates of B lymphocytes within the CALT appeared more frequently as the chicken aged and as the germinal centers developed. All T lymphocyte subsets stained within the CALT surrounded the germinal centers and increased in concentration between 1 and 4 weeks of age;Both of these techniques were used to evaluate lymphocytes from the HALTs of chicks intraocularly inoculated with Newcastle disease virus (NDV). Velogenic and lentogenic strains of NDV were used. In the in vitro lymphoproliferation assay, Harderian gland T cells isolated from the NDV inoculated chicks had a significantly higher lymphoproliferative response to in vitro NDV antigen compared with the T cells isolated from phosphate buffered saline control hatch mates. A significant increase in all T lymphocyte subsets was observed in the in situ analysis of the Harderian glands from chicks inoculated with either lentogenic or velogenic NDV when compared with chicks inoculated with phosphate buffered saline solution. No differences in the lymphocyte subsets were observed from the CALT collected from any of the three treatment groups. The results presented could contribute to vaccine program development against respiratory diseases.