Invariant natural killer T cells (iNKT) are a population of innate-like T lymphocytes with crucial roles in protective responses to infectious agents, particularly Streptococcus pneumoniae. Despite their abundance in the lungs, there is limited understanding of how iNKT cells mediate protection there. Prior work from our group showed after pulmonary S. pneumoniae infection, iNKT cells are rapidly activated in a CD1d-dependent manner. Similar to CD4+ T cells, iNKT cells have been sub-classified into NKT1, 2, and 17 subsets. Here we investigated the roles of these subsets in protection from S. pneumoniae. In uninfected mouse lungs, NKT1 and NKT17 cells were the predominant iNKT subsets, with most NKT17 cells in the tissue and NKT1 cells mainly within the vasculature. We utilized fluorescent bacteria, flow cytometry, and intravital microscopy to analyze the lungs during infection. By 24 hours, the bacteria localized within the tissue, and were taken up by alveolar macrophages, neutrophils, and to a lesser extent DCs. As early as 14 hours, most of the lung NKT17 cells produced IL-17, while only a minor percentage of NKT1 cells produced IFN-γ. This correlated with data from Nur77^GFP reporter mice that indicate TCR-mediated activation. At 24 hours, approximately 75% of NKT17 but only 30% of NKT1 cells were TCR-activated; suggesting NKT17 cells may play a dominant role early in infection. Yet, at the same time, NKT1 cells expanded to become the major subset in the lungs, suggesting they also could be relevant for protection. In summary, our data show not only differential localization of iNKT cell subsets in the lung, but also differences in the timing of their TCR-mediated activation, suggesting the NKT17 subset could be particularly important for protection. Experiments are underway to determine which cell type(s) presents antigen, whether iNKT cells produce other cytokines, and the consequences of removing individual subsets.