Hepatitis B virus (HBV) core antigen (HBcAg) is a highly immunogenic subviral particle. Here, we review recent progress in the use of HBcAg as a carrier moiety for heterologous epitopes. To define surface exposed and immunogenic insertion sites for foreign epitopes in HBcAg, peptidic epitopes representing binding sites for virus neutralizing antibodies on the HBV surface antigens were inserted at different positions within HBcAg using genetic engineering in an Escherichia coli expression system (Schödel et al. (1992) J. Virol. 66, 106-114). While fusion to the N-terminus required a linker to become surface accessible, both fusion to the N-terminus and to the C-terminus was compatible with particle assembly and preserved the native antigenicity and immunogenicity of HBcAg. Fusion to an immunodominant internal site of HBcAg reduced the HBcAg immunogenicity and antigenicity and most drastically enhanced the immunogenicity of the inserted foreign epitope. This internal site of HBcAg was used to express circumsporozoite antigen (CS) repeat epitopes of two rodent malaria parasites and of Plasmodium falciparum (Schödel et al. (1994b) J. Exp. Med. 180, 1037-1046 and Schödel et al. (1995a) 95th ASM General Meeting, Washington DC, Abstr. E61). When purified from recombinant Salmonella typhimurium, the hybrid HBcAg-CS proteins were particulate and displayed CS antigenicity as well as reduced HBc antigenicity, as compared to native HBcAg. Immunization of several mouse strains with HBcAg-CS hybrid particles resulted in high titered serum anti-CS antibodies representing all murine IgG isotypes. Immunization of mice with HBcAg or HBcAg-CS particles formulated on alum, complete Freunds or incomplete Freunds adjuvant resulted in equivalent anti-CS and anti-HBc serum antibody titres. The possible influence of carrier-specific immunosuppression was examined and pre-existing immunity to HBcAg did not significantly alter the immunogenicity of hybrid HBcAg particles suggesting that they would be useful carrier moieties for repeated immunizations against multiple haptens or in immune subjects after HBV infection. Examination of T cell recognition of HBcAg-CS particles revealed that HBcAg-specific T cells were universally primed and CS-specific T cells were primed if the insert contained a CS-specific T cell recognition site. This indicates that the internal amino acid position in HBcAg is permissive for the inclusion of heterologous functional T helper as well as B cell epitopes. BALB/c mice immunized with HBcAg-CS1 were protected against P. berghei challenge to 90% and 100%, respectively, in two independent experiments.