A DNA vaccine (VCL1010/MAP1) containing the major antigenic protein 1 (MAP1) gene of Cowdria ruminantium, driven by the human cytomegalovirus (HCMV) enhancer-promoter, was injected intramuscularly into 8-10 week-old female DBA/2 mice after treating them with 50 microliters/muscle of 0.5% bupivacaine three days previously. Up to 75% of the immunized mice seroconverted and reacted with C. ruminantium antigen blots. Splenocytes from immunized mice, but not from control mice, proliferated in response to the recombinant MAP1 and to C. ruminantium antigens in in vitro lymphocyte proliferation tests. These proliferating cells secreted IFN-gamma and IL-2 at concentrations ranging from 610 pg/ml to 1290 pg/ml and from 152 pg/ml to 310 pg/ml, respectively. Only up to 45 pg/ml and 42 pg/ml of IFN-gamma and IL-2, respectively, were detected in supernatants of splenocytes from control mice. In experiments testing different VCL1010/MAP1 DNA vaccine dose regimens (25-100 micrograms/dose, two or four immunizations), survival rates of 23% to 88% (35/92 survivors/total in all VCL1010/MAP1 immunized groups) were observed on challenge with a lethal dose of cell culture-derived C. ruminantium organisms. In contrast, survival rates of 0% to 3% (1/144 survivors/total in all control groups) were recorded for control mice. This study demonstrates that MAP1 is a protective antigen and validates the concept of DNA vaccines against heartwater.