Spore-forming recombinant Bacillus subtilis 1A289 containing plasmid pC194 carrying an alpha-amylase encoding gene was used for the production of the cloned gene product in batch culture. In flask culture, the alpha-amylase activity at a culture temperature of 45 degrees C, was 643 U/ml which was 2-fold higher than that at 37 degrees C. No spore formation was detected in the culture. When the cells were cultured in a jar fermenter at 45 degrees C with a dissolved oxygen (DO) level of 90%, the enzyme activity was only 184 U/ml which was only 30% of that in the flask culture. However, when the DO level was controlled at 5%, the activity was 846 U/ml. In the case of cultures at 45 degrees C with a DO level higher than 50%, spore concentration and spore fraction were more than 3.3 x 10(8) spores/ml and 85-100%, respectively. These values were 10 times higher with respect to spore concentration and 6 times higher with respect to the fraction of cells forming spores than those at a DO level of 5%. There were three types of cells in the fermenter : cells resistant to chloramphenicol and producing alpha-amylase; cells having lost plasmid pC194; cells forming spores. The specific enzyme activity of vegetative cells was 1.9 kU/mg dry cell weight, while that of spores was 0.02 kU/mg dry cell weight. The alpha-amylase gene expression of the spore-forming recombinant B. subtilis was affected strongly by culture temperature, DO level, and fraction of cells forming spores.