Aims: To determine effects of inner membrane lipid composition on Bacillus subtilis sporulation and spore proper-ties. Methods and Results: The absence of genes encoding lipid biosynthetic enzymes had no effect on B. subtilis sporulation, although the expected lipids were absent from spores' inner membrane. The rate of spore germination with nutrients was decreased c. 50% with mutants that lacked the major cardiolipin (CL) synthase and another enzyme for synthesis of a major phospholipid. Spores lacking the minor CL synthase or an enzyme essential for glycolipid synthesis exhibited 50-150% increases in rates of dodecylamine germination, while spores lacking enzymes for phosphatidylethanolamine (PE), phosphatidylserine (PS) and lysylphosphatidylglycerol (I-PG) synthesis exhibited a 30-50% decrease. Spore sensitivity to H2O2 and tert-butylhydroperoxide was increased 30-60% in the absence of the. major CL synthase, but these spores' sensitivity to NaOCl or Oxone (TM) was unaffected. Spores of lipid synthesis mutants were less resistant to wet heat, with spores lacking enzymes for PE, PS or I-PG synthesis exhibiting a two to threefold decrease and spores of other strains exhibiting a four to 10-fold decrease. The decrease in spore wet heat resistance correlated with an increase in core water content. Conclusions: Changing the lipid composition of the B. subtilis inner membrane did not affect sporulation, although modest effects on spore germination and wet heat and oxidizing agent sensitivity were observed, especially when multiple lipids were absent. The increases in rates of dodecylamine germination were likely due to increased ability of this compound to interact with the spore's inner membrane in the absence of some CL and glycolipids. The effects on spore wet heat sensitivity are likely indirect, because they were correlated with changes in core water content. Significance and Impact of the Study: The results of this study provide insight into roles of inner membrane lipids in spore properties.