His-Asp phosphorelays are widespread signal transduction mechanisms in bacteria, fungi, and higher plants. In order to investigate a His-Asp phosphorelay network in filamentous fungi, which has been genetically characterized in part, we attempted to construct an in vitro phosphotransfer network in Aspergillus nidulans comprising all the necessary components. As a first step, we established an in vitro phosphotransfer system with a histidine-containing phosphotransmitter YpdA, a response regulator SrrA, and a bacterial histidine kinase ArcB as a phosphate donor. We demonstrated the phosphotransfer from ArcB to A. nidulans YpdA and the subsequent transfer from YpdA to SrrA. This is the first direct biochemical evidence for the presence of the phosphotransfer system in filamentous fungi. Furthermore, a retrograde phosphorylation from YpdA to FphA, a histidine kinase similar to bacterial phytochrome, was found. The overall picture of the His-Asp phosphorelays in A. nidulans is discussed based on the results of the in vitro study.