l-Alanine initiates the germination of spores of Bacillus subtilis by entering two metabolic pathways. The products of one pathway, which is inhibited by d-alanine or by elevated temperature, can also be derived from a combination of fructose, glucose, and K(+). The present study demonstrated that the products of the other pathway can be derived from l-asparagine or l-glutamine or, to a lesser extent, from several other amino acids. Hence, the combination of l-asparagine (or l-glutamine), fructose, glucose, and K(+) can initiate spore germination in the absence of l-alanine. Spores preincubated in a combination of asparagine and fructose do not lose refractility, optical density, or heat resistance, and do not take up methylene blue stain. The spores do, however, undergo some reaction which prepares them for a more rapid response to the later addition of glucose and K(+). This preincubation reaction has an optimal temperature of about 44 C.