Two-dimensional TLC analysis of 32P-labeled nucleotides extracted from the yeast Saccharomyces cerevisiae reveals that two highly phosphorylated nucleotides are synthesized during sporulation. These nucleotides have been identified as adenosine 5'-tetraphosphate (ppppA) and adenosine 5'-pentaphosphate (pppppA). The synthesis of ppppA and pppppA commences late in sporulation and follows formation of ascospores. The maximum concentration of ppppA and pppppA in sporulating yeast cultures was 2% and 1.5%, respectively, that of ATP. Adenosine 5'-tetraphosphate and 5'-pentaphosphate are unique to this stage of yeast development and are absent in vegetative yeast cells. Since these nucleotides are also absent in asporogenous a/a and alpha/alpha cells, it is reasonable to propose that they are signal nucleotides marking one of the stages of yeast development--i.e., ascospore formation.