Differences in immune responses of human mononuclear leukocytes (MNL) have been demonstrated following exposure in vitro to influenza virus or respiratory syncytial virus (RSV). In the current studies, we sought to identify early differences in reactive subpopulations that emerge from within the heterogeneous resting MNL pool after challenge. MNL were sham-exposed or exposed to influenza virus or RSV, separated, and retrieved by countercurrent centrifugal elutriation after 3 d. Exposure to influenza virus caused a relative decline in the number of large MNL, but an increase in small lymphocytes. Large cells that remained included primitive lymphoblasts, rare plasma cells, and typical lymphocytes of progressively greater volume. Exposure to RSV increased the number of large MNL, but diminished the number of small lymphocytes. The subpopulation of large cells consisted of atypical and large granular lymphocytes. Furthermore, deletion of the latter large, reactive lymphocytes led to abrogation of an RSV-specific proliferative response upon subsequent challenge. Thus, the specific and different subpopulations reactive after infectious virus challenge could be identified, retrieved, and manipulated without dependence on a priori, phenotypic markers.