Tetanus toxoid is a routine positive control antigen for cellular assays. Previous studies identified multiple tetanus toxin (TT) epitopes, including some 'universal' epitopes. However, rigorous HLA-restricted study of tetanus toxoid responses is still lacking. In this study, the tetramer-guided epitope mapping approach was used to identify CD4+ T-cell epitopes within the TT heavy chain restricted by 10 different class II alleles. Of 106 peptides tested, 52 contained epitopes. Response frequencies toward specific epitopes varied, indicating prevalent and rare specificities. Most antigenic peptides (85%) were presented by one or two class II alleles. For peptides presented by three or more alleles, truncation studies revealed that some contained multiple epitopes. These results contrast with the perceived notion that tetanus toxoid responses are dominated by universal CD4+ T-cell epitopes. Rather these results illustrate heterogeneous T-cell responses for different class II alleles and individual-specific variation of the T-cell repertoire.