The causative agent of heartwater, Ehrlichia ruminantium, is a tick-transmitted pathogen that infects bovine endothelial cells. Due to the obligate intracellular nature of this organism obtaining pure material in sufficient quantities for challenge studies is difficult. A murine model is frequently used to study potential vaccine candidates but giving reproducible challenges in this model for heartwater has always been problematic. We have therefore performed a series of experiments to optimize the parameters governing the reproducibility of challenge material. Two cryoprotectants were compared for the preparation of challenge material, buffered lactose peptone (BLP) and sucrose-potassium-glutamate (SPG). In addition two sources of virulent E. ruminantium were used, infected bovine endothelial cultures and infected mouse spleen homogenates. We also examined practical parameters affecting the reproducibility of challenge experiments: the time it takes to deliver the challenge material, the length of time a mouse remains immune to E. ruminantium challenge, and the effect of a given challenge dose. Finally, we performed a pilot study to determine whether mice could be used to titrate challenge material to be used for experiments in sheep. We found that: (a) E. ruminantium-infected mouse spleen homogenate provides more reproducible challenges than tissue culture material; (b) SPG is a better cryoprotectant than BLP; (c) challenge material should be used within 20min of thawing; (d) it is not essential to use syngeneic material for murine challenge experiments; (e) Balb/c mice are more sensitive to E. ruminantium challenge than C57BL/6J mice; (f) mice immunized by infection and treatment for use as positive immune controls should be challenged within 3 months of immunization; and (g) mice should be challenged with a dose not exceeding 10 LD(50)s.