Splenocytes from non-immune mice were stimulated in vitro using a kit of cytokine preparations (obtained from murine MLR and EL-4 cell cultures), and concomitantly immunized with African swine fever (ASF) virus antigen. In addition, fusions were performed at 5 days after primary or secondary stimulation/immunization. The detection of specific antibodies in the culture supernatants was not successful. In contrast, specific antibody-producing hybridomas could be generated, and this was at least comparable to a standard in vivo immunization regime, even though the optimum fusion ratio employed with these in vitro immunized splenocytes was one which is not optimum when in vivo immunized lymphocytes are used. Consequently, it would appear that hybridoma generation is a more sensitive method than the direct measurement of antibody at detecting in vitro primary immune responses. After primary in vitro immunization, the majority of immunoglobulins produced were apparently of the IgG isotype, with only 8-17% clearly IgM. These antibodies were mainly against VP73 (the major viral envelope protein) as expressed on viral antigen extracted from infected cells, although other specificities were also found. This demonstrated, by in vitro means, that the VP73 carried dominant immunogenic epitopes on ASF virus. Such observations show that the in vitro responses were closely related to those which have been detected in vivo.