To study the reactivity of nasal-associated lymphoid tissue (NALT) and its position in the mucosal immune system, rats were intranasally challenged with 200 micrograms TNP-LPS. Priming had occurred 15 days previous to the challenge with the same antigen and dose, either intranasally, intratracheally, subcutaneously in the cheek or intraperitoneally. The number of anti-TNP antibody-forming cells (AFC) was determined in various tissues using the conjugate TNP/alkaline phosphatase. Generally, anti-TNP AFC were predominantly found in the posterior cervical lymph nodes, while NALT contained hardly any such AFC. The highest response in the posterior cervical lymph nodes occurred on day 5, after subcutaneous priming and intranasal boosting. This also evoked peak responses in several other tissues. The highest response in spleen and lung occurred on day 7 after intraperitoneal priming and intranasal boosting. Irrespective of the immunization route, IgA was the least produced isotype in the spleen as compared to antigen-specific IgG and IgM. In the posterior cervical lymph nodes, besides specific IgG and IgM, a considerable proportion of specific IgA was produced. All four immunization routes yielded anti-TNP antibodies in serum. As for the non-lymphoid cells, the intratracheal-intranasal immunization protocol induced an increase in pulmonary macrophages on days 3 and 5. The immunological role of lung macrophages is discussed.