Mouse models and a recent vaccine trial have indicated the importance of T-cell immunity to the circumsporozoite protein (CSP) of malaria sporozoites. One of the major impediments for the development of a CSP-based vaccine is that human T-cell epitopes, identified on the CSP, span regions of significant point mutational polymorphism. Studies with human and mouse T-cell clones have indicated that this polymorphism affects T-cell cross-reactivity to Th2R and Th3R, the two most polymorphic and immunodominant epitopes. We extend this observation with polyclonal human T-cell lines, from 11 donors, raised to known variants of Th2R. These lines showed limited but variable cross-reactivity with the heterologous peptides. T cells from B10.A4(R) (I-Ak) mice immunized with each of 18 natural variants of Th2R indicated a similar, limited, cross-reactivity. I-Ak competition assays showed that a number of peptides were unable to bind because of a single polymorphic residue. In both the human and mouse assays, analysis of the sequences of immunogenic cross-reactive and non-cross-reactive peptides suggested that the individual polymorphic residues affect the three-dimensional conformation of the peptide within the major histocompatibility complex (MHC) groove in an, as yet, unpredictable way. These observations argue that design of an epitope able to generate broad cross-reactivity is, to date, not possible. However, despite the limited cross-reactivity of the individual human T-cell lines, most of the donors had T-cell repertoires capable of recognizing all or nearly all of the variants tested, which supports a strategy using a multivalent vaccine.