We previously described that an inactivated vaccine against heartwater prepared from Cowdria ruminantium (Crystal Springs strain) organisms and administered in complete Freund's adjuvant (cFA) protected sheep against homologous needle challenge. Further studies, described herein, demonstrated that this vaccine protected 100% of sheep against death on challenge with laboratory-infected ticks and with field ticks in a heartwater endemic farm, whereas a mortality rate of 44% and 62%, respectively, was recorded in the control sheep. Subsequently, the Mbizi strain of C. ruminantium was incorporated into the vaccine because of its wider cross-protective capacity, and trial data suggested that protection may be achieved against challenge with diverse geographical strains using this strain. The efficacy of five adjuvants with acceptable safety was compared with that of cFA. Against a homologous intravenous challenge, highest survival rates were observed in sheep vaccinated with inactivated C. ruminantium in either cFA, Montanide ISA 50 or Quil A. The vaccine prepared in Montanide ISA 50 protected six of seven sheep against natural challenge from field ticks on a farm in Zimbabwe where heartwater is endemic, whereas six of seven control sheep died (P = 0.029). These data support optimization of the vaccine prepared in Montanide ISA 50, followed by evaluation of its efficacy in all target domestic ruminant species and in other geographical regions where heartwater constrains livestock production.