MHC class II molecules associate with invariant chain (li) during biosynthesis. li facilitates folding of class II molecules, interferes with their association with peptides, and is involved in their transport. The murine li gene encodes two chains, p31 and p41. The role of these isoforms has been studied in vitro only in inappropriate antigen-presenting cells. To circumvent this problem, we have generated invariant chain-deficient mice (Δli), which express exclusively the p31 and p41 isoforms. Low level expression of p31 or p41 is not sufficient for rescuing high levels of cell surface class II expression. However, low levels of the typical compact dimer conformation indicative of tight peptide binding are observed. Thus, both isoforms participate in class II folding and assembly. Furthermore, p31 and p41 retrieve the CD4⁺ T cell population, which is reduced in the (Δli) mice. Moreover, the immune response to protein antigen is restored by both isoforms.