BALB/c mice were inoculated intraperitoneally either once only, or up to four times at weekly intervals, with viable Rickettsia rickettsii, Rickettsia conorii or the Israeli spotted fever group rickettsia. Sera collected one week after the last inoculation were tested for the presence of antibodies reactive with the above organisms by indirect fluorescent antibody testing and Western blot. With repeated inoculations there was a general progressive rise in homologous and heterologous immunofluorescence titers although the increase after the first inoculation was always the greatest. For each rickettsia, the homologous titers were higher than the heterologous titers. Western blots showed that the reactive antibodies were against rickettsial high molecular mass species specific protein antigens and homologous species-specific antibody reactions were detectable earlier than heterologous cross-reacting antibody reactions. Antibodies in mice sera did not react with the group specific lipopolysaccharide-like antigens of the rickettsiae although such reactivity was strong in Western blots with sera from patients suffering from acute Rickettsia conorii infections. Our findings suggest that the intraperitoneal route of inoculation of BALB/c mice can be used for the differentiation of spotted fever group rickettsiae.