Abstract We developed a sensitive competitive inhibition radioimmunoassay able to trace pmoles of a defined peptide eluted from major histocompatibility complex (MHC) class II molecules that were subsequently fractionated by RP-HPLC. In this assay we used a model synthetic peptide corresponding to amino acid residues 110–120 from the hemagglutinin (HA) of PR8 influenza virus, and affinity purified rabbit antibodies specific for this peptide. The HA110–120 peptide binds to I-E d class II molecules on the surface of APCs and is recognized by specific CD4 + T helper cells. 2PK3 B lymphoma cells (H-2 d) were pulsed with HA110–120 peptide or PR8 virus, lysed, the MHC class II molecules extracted, and bound peptides eluted. After separation by RP-HPLC, the fractions were tested for inhibition of the binding of rabbit anti-HA110–120 antibodies to peptide coated microtiter plates. A significant inhibitory activity was observed with one peak when the cells were pulsed with HA110–120 peptide and two peaks when pulsed with PR8 virus. The inhibitory activity was correlated with the presence of HA110–120 peptide as demonstrated by peptide sequencing. The assay is reproducible and sensitive to 1 pmol of antigenic peptide. This assay can be useful to identify microbial peptides with defined structure and antigenicity among the multiple peptides bound to class II molecules.