A general protocol was derived for optimizing the production of pure, high concentration Clostridium endospore suspensions. Two sporulation methods were developed that yielded high concentrations of notably pure Clostridium sporogenes, C. hungatei and C. GSA-1 (Greenland ice core isolate) spore suspensions (10 ml of 10(9) spores ml(-1) with > 99% purity each). Each method was derived by evaluating combinations of three sporulation conditions, including freeze drying of inocula, heat shock treatment of cultures, and subsequent incubation at suboptimal temperatures that yielded the highest percentage of sporulation. Pure spore suspensions were characterized in terms of dipicolinic acid content, culturability, decimal reduction time (D) value for heat inactivation (100 degrees C) and hydrophobicity. While some Clostridium species produce a high percentage of spores with heat shock treatment and suboptimal temperature incubation, other species require the additional step of freeze drying the inocula to achieve a high percentage of sporulation. Pure Clostridium spore suspensions are required for investigating species of medical and environmental importance. Defining the conditions for optimal spore production also provides insight into the underlying mechanisms of Clostridium sporulation.