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MHC tetramers have proven to be powerful reagents for the analysis of MHC class I-restricted T cells. However, generating similarly reliable reagents for MHC class II-restricted T cells has been elusive. Here we evaluated the utility of MHC class II:γ2aFc multimers, which contain the MHC class II extracellular domains, with or without recombinantly attached peptides, dimerized via a fos–jun leucine zipper and attached to the hinge of murine IgG2a. We have successfully generated 24 multimers in either myeloma or Drosophila melanogaster S2 cells, with an average yield of 7 mg/L. ‘Empty’ MHC class II:γ2aFc multimers were effectively used in peptide binding assays. Similar versions that contained recombinantly attached peptides stimulated T cells in an antigen-specific, MHC-restricted manner, and identified antigen-specific naı¨ve and effector T cells by flow cytometry. Furthermore, we have successfully used these reagents to stain T cells generated following viral infection. Thus, MHC class II:γ2aFc multimers are robust and reliable reagents for the analysis of MHC class II-restricted T cells. [Copyright &y& Elsevier]