We have investigated the immunogenicity of defined sequences of the circumsporozoite (CS) protein of the murine malaria parasite, Plasmodium yoelii. A 21-ner synthetic peptide from the nonrepetitive region of the CS protein (position 59-79, referred to as Py1) induced T cell proliferative responses in H-2d and, to a lesser extent, in H-2b mice. Conversely, a synthetic peptide (referred to as Py4) consisting of four (QGPGAP) repeats of the P. yoelii CS protein, induced an antibody response only in H-2b mice. No antibody response was observed when the Py3 peptide, consisting of three (QGPGAP) repeats, was used as an immunogen. When cross-linked to the Py4 repetitive peptide, the Py1 sequence behaved as a T helper epitope allowing the production of anti-Py4 antibodies in H-2d mice. Several long-term T cell lines and clones specific for the nonrepetitive Py1 peptide were originated in vitro from both H-2d and H-2b mice. These lines and clones were CD4+ and proliferated in a major histocompatibility complex-restricted fashion. Furthermore, Py1-specific T cell lines and clones did not proliferate in the presence of synthetic peptides from an analogous region of another rodent malaria parasite, P. berghei, despite the high degree of homology existing in this sequence of the two CS proteins. Finally, supernatants from 7 out of 13 clones (from BALB/c mice) produced detectable amounts of interleukin 2 and interferon-gamma; whereas supernatants from the 4 clones from C57BL/6 and 2 from BALB/c mice contained detectable amounts of interleukin 5. These results show that functionally heterogenous CD4+ T cell populations, belonging to either TH1 or TH2 subset, are activated upon immunization of mice with the P. yoelii Py1 synthetic peptide. It is not yet known what differential role these CD4+ subsets play during the malaria infection or after immunization with different malaria T cell epitopes. This knowledge may have a particular impact in the design of effective subunit vaccines against malaria.