We previously described differences in the 50% protective dose and isotype-specific antibody secreting cell (ASC) responses to US and Russian influenza A cold-adapted (ca) donor strains in the lungs of BALB/c mice [Wareing MD, Watson JM, Brooks MJ, Tannock GA. Immunogenic and isotype-specific responses to Russian and US cold-adapted influenza A vaccine donor strains A/Leningrad/134/17/57, A/Leningrad/134/47/57, and A/Ann Arbor/6/60 (H2N2) in mice. J Med Virol 2001;65(1):171-7]. A/Leningrad/134/17/57(Len/17-ca) was shown to be a superior immunogen to A/Leningrad/134/47/57-ca (Len/47-ca), which, in turn, was superior to A/Ann Arbor/6/60-ca (AA-ca) but no other comparative data exist. In order to extend our findings and determine a means for selecting the most immunogenic ca influenza A vaccine, the intracellular cytokine responses by CD4+ and CD8+ T cells to AA-ca, Len/47-ca and Len/17-ca and their respective wild-type parental viruses were compared in mice. Day 5 after infection with Len/17-ca, when levels of IL-2, -4 and -10 were highest in the mediastinal lymph nodes (MLN) and lungs, was chosen as the optimum time to harvest lymphocytes and 72 h was determined to be the optimum re-stimulation period for lymphocytes by APCs. Under these conditions, the frequency of CD4+ and CD8+ cells expressing cytokines was highest in the lungs compared with the MLN. A dominant IL-6 response was induced, although all virus strains induced a Th1/Th2 cytokine profile. While the CD8+ cytokine response appeared non-specific, the cytokine response elicited in the lungs by CD4+ cells to Len/17-ca-inoculation was greater than that induced by Len/47-ca, or AA/ca. The CD4+ cytokine response in the lungs may be a useful measure of immunogenicity to determine the most effective influenza reassortant for inclusion in vaccines.