The importance of in situ lymphocyte proliferation for net accumulation of lung lymphocytes during pulmonary immune responses and in immunologic lung diseases remains uncertain. Accordingly, we studied the experimental pulmonary immune response of antigen-primed C57BL/6 mice to intratracheal challenge with the particulate antigen sheep red blood cells. Uptake of nucleotide analogs (bromodeoxyuridine in vivo and tritiated thymidine in vitro), expression of the cell activation antigens CD25 and CD69 by flow cytometry, and response to the antimitotic agent hydroxyurea (in vivo) were measured. Although many lung lymphocytes and CD4+ T cells were CD25+ and CD69+, indicating recent activation, all techniques demonstrated that lung lymphocytes proliferated minimally in vivo. Blockade of cell division by hydroxyurea administration for 24 h did not significantly decrease lung lymphocyte accumulation on Day 3 after challenge. Lung lymphocytes also proliferated minimally in vitro (even on macrophage removal and despite addition of exogenous interleukin [IL]-2 or IL-4). However, lung lymphocytes responded vigorously to mitogens (immobilized anti-CD3, phytohemagglutinin, or concanavalin A), excluding global unresponsiveness to restimulation. Thus, in this model of pulmonary immunity, accumulation of lung lymphocytes does not require local T-cell proliferation and presumably depends instead on recruitment.