In a previous paper, El-Bisi and Ordal (1956a) reported that increased levels of phosphate, in the growth and sporulation medium, significantly reduced the thermal resistance of spores of Bacillus coagulans var. thermoacidurans. It was postulated that the phosphate anion lessened the availability of divalent cations to the sporulating cells and that spores so produced possessed a reduced thermal resistance. A relationship between the calcium content and the degree of thermal resistance of bacterial spores has been suggested by Curran (1952) and by Sugiyama (1951). Williams (1929) reported that the addition of magnesium to peptone containing media yielded spores of Bacillus subtilis of increased thermal resistance. In this investigation, the effect of the divalent cations, calcium, magnesium, and manganese, added to the sporulation medium, was studied in more detail for their effect on the thermal resistance of spores of B. coagulans var. thermoacidurans. MATERIALS AND METHODS Test culture. B. coagulans var. thermoacidurans, American Type Culture Collection no. 8038, was used in this investigation. Stock cultures were maintained on thermoacidurans agar (Difco) containing additional 1 ppm MnSO4. Media. Spore crops were produced either on agar surface cultures or in broth shake cultures. Basal media used were thermoacidurans agar (proteose-peptone, 0.5 per cent; yeast-extract, 0.5 per cent; glucose, 0.5 per cent; K2HPO4, 0.5 per cent; and agar, 2 per cent) and thermoacidurans broth (the same components omitting the agar).