Reverse genetics was used to modify the influenza virus genome by inserting the p46-63 sequence of hen egg lysozyme (HEL) into the neuraminidase stalk of the virus. The resulting virus, HEL-Flu, contained the epitopes recognized by CD4+ T cells from 3A9-TCR transgenic mice (C3HTg). Here, we show that HEL-Flu was infectious in the respiratory tract of both C3H and C3HTg mice, the latter animals showing an early, transient morbidity. Splenic dendritic cells and certain cloned populations of splenic macrophages and brain microglia constitutively presented infectious and inactivated HEL-Flu to the T cells in an Ag-specific and MHC class II-restricted manner. These results demonstrate the utility of HEL-Flu in assessing the APC activity for naive T cells; they also extend the previous studies showing that discrete populations of macrophages and microglia constitutively process and present Ag to naive T cells.