An assessment of T-cell-mediated immune functions (i.e., lymphocyte proliferation assay) in the chicken, unlike the determination of antibody levels, is not routinely performed. This is primarily because of difficulties in the isolation of relatively pure populations of lymphocytes and the use of radioactive isotopes. To address these issues, the goals of our study were to optimize a method for isolating and enriching avian lymphocyte populations and to develop a nonradioactive lymphocyte proliferation assay. To accomplish these goals, we used a multiple slow-speed centrifugation technique combined with a "swirl" collection technique for lymphocyte isolation from chicken peripheral blood. After a fraction enriched with lymphocytes was obtained, a simple, rapid colorimetric and fluorometric assay (lympho-pro) to indirectly determine mitogen-induced proliferation was adapted and compared with the "Gold Standard" [3H]thymidine. Chickens of different ages and two genetic strains were used in this study. Lymphocytes were stimulated with various concentrations of concanavalin A (Con A, T-cell mitogen) or phorbol 12-myristate 13-acetate + ionomycin (pan lymphocyte mitogen). Our studies showed that the pattern of lymphocyte proliferation assessed by the Alamar blue-based lympho-pro assay was similar to the [3H]thymidine incorporation assay. Younger birds had higher levels of mitogen-induced proliferation when compared with adults of the same genetic strain. Because the lympho-pro assay, unlike [3H]thymidine, does not require lysis of cells to assess proliferation, cells that have undergone stimulation/proliferation can be subsequently characterized by staining with antibodies against cell surface antigens and analysis by flow cytometry. Another notable advantage of the lympho-pro assay is the rapidity of assessment and nontoxicity. In conclusion, this assay may be of value in assessing some aspects of T-cell-mediated immunity in both avian research and avian medicine diagnostic settings.