Aspergillus niger van Tieghem was cultured by the method of synchronous and submerged culture. Throughout the culture, sporulation was occured. Highly phosphorylated nucleotides in sporulating mycelia were detected to assure whether the eucaryotic Aspergillus niger produce these substances or not during the differentiation. Phosphorylated nucleotides were extracted from the conidiophore, bearing mycelia and spore forming body, these nucleotides were identified by TLC with P.E.I. cellulose. Guanosine tetraphosphate was found in both phialide forming mycelia and spore forming body. The contents of free amino acids were assayed and its level was found to increase at early stage of sporulation. The effects of 8-azaguanine examined, it was found to prevent spore formation and to made abnormal structure. The effects of inosinic monophosphate and guanosine monophosphate on spore formation were examined, spore formation was enhanced by these nucleotides.