Unusual highly phosphorylated nucleotides are found in sporulating cells of Bacillus subtilis. Adenosine 3'(2')-diphosphate 5'-diphosphate, ppApp (highly phosphorylated nucleotide I), and adenosine 3(2')-dephosphate 5'-triphosphate, pppApp (highllls are starved for carbon and nitrogen sources. These nucleotides are correlated with sporulation because only ribosomes from sporulating but not vegetative cells are able to synthesize ppApp and pppApp in vitro. Two other nucleotides, adenosine 3'(2')-triphosphate 5'-triphosphate, pppAppp (highly phosphorylated nucleotide IV), and a nucleotide with a tentative structure of ppZpUp (highly phosphorylated nucleotide III), where Z is an undetermined sugar, also seem to be involved in regulation of sporulation, especially initiation of sporulation. Sporulation can be initiated even in the presence of amino acids, salts, vitamins etc. in logarithmically growing or stationary-phase cells when carbon sources, i.g. glucose, are used up or artifically removed from the medium. A drastic increase in spore titer is observed 4--5 h later. Also, carbon starvation causes accumulation of the highly phosphorylated nucleotides pppAppp and ppZpUp. On the other hand, sporulation is prevented under the same conditions when excess glucose is maintained in the medium. Correlated with this inhibition of sporulation is the inhibition of formation of highly phosphorylated nucleotides, pppAppp and ppZpUp. Since synthesis of these nucleotides is closely related to sporulation, we anticipate that these substances can cause initiation of development in B. subtilis. Further evidence for our hypothesis on initiation of sporulation by highly phosphorylated nucleotides is that phosphate starvation also causes sporulation with prior accumulation of pppAppp and ppZpUp. Apparently, as long as phosphate is present to synthesize phosphorylated metabolites of glucose, formation of highly phosphorylated nucleotides is repressed. Derepression occurs when either lack of glucose or phosphate or both prevents synthesis of phosphorylated metabolites of glucose allowing synthesis of highly phosphorylated nucleotides. These nucleotides, representing the signal 'lack of glucose or phosphate', then somehow cause changes in gene activity, initiating the complex process of sporulation. Whether or not pppAppp alone or together wtih ppZpUp or even further substances (nucleotides, proteins etc.) is necessary for the above described processes will be answered with the help of suitable mutants lacking the ability to synthesize either one or both regulatory nucleotides. Guanosine 3'(2')-diphosphate 5'-diphosphate, ppGpp, and guanosine 3'(2')-diphosphate 5'-triphosphate, pppGpp, are not involved in regulation of devlopment as is shown by using a normally sporulating mutant of B. subtilis, unable to synthesize these nucleotides.