The contribution of viral infectivity to the expression of MHC class II-restricted T cell determinants was studied. A murine I-Ed-restricted T cell hybridoma recognizing the neuraminidase (NA) glycoprotein of influenza PR8 virus was stimulated strongly by infectious virus but failed to recognize antigen introduced on noninfectious virions. Recognition correlated with the de novo synthesis of viral NA within infected APC. The effectiveness of infectious virus did not depend strictly upon the amount of NA present in cultures, since high NA concentrations could be achieved by addition of nonreplicative virus without being stimulatory for NA-specific T cells. Recognition of a determinant generated only when synthesized in murine host cells was ruled out, since, in high concentration, NA isolated from purified egg-grown virions, even if reduced and alkylated, was recognized by the T hybridoma clone. Isolated NA was recognized when added to pre-fixed APC, suggesting that this form of antigen was able to bypass the usual processing pathway of exogenous proteins. Data suggest that endogenously synthesized antigen may contribute most significantly to presentation of labile T cell determinants. In addition to NA, recognition of an I-Ed-restricted determinant of the influenza hemagglutinin (HA) molecule, shown previously to have a relatively short half-life on APC surfaces, was enhanced greatly by infectious virus. In contrast, T cell recognition of a more stably expressed I-Ed-restricted site of the same HA polypeptide was only marginally improved on infected APC.