After more than a hundred years of archaeological explorations and studies in Iran, for various reasons, the eastern regions have received less or no attention from archaeologists, and there is very limited publication of archaeological activities in this part of Iran. The ancient site of Kalehkoob Aisk is one of the limited prehistoric sites in South Khorasan province and in general in eastern Iran, which has enough cultural deposits for the purpose of providing a chronology and understanding the prehistoric cultural sequence of this region. The excavations carried out at this site led to the identification of previously unknown prehistoric cultures in the region. The most significant achievement of the excavation at this site is the identification of fourth millennium BC cultures with a southwestern and Mesopotamian origin, the evidence of which is found far from the center in this area. These cultural evidence, which can be considered contemporary with the Shush II or New Uruk cultures, include various types of characteristic pottery of this period such as incised edged pottery, coarse ware dishes, tubular vessels, and handle-faced beakers, as well as decorated pottery and delicate wheel-made pottery that are known in southwestern, western, northwestern, southeastern, and central plateau of Iran, but are identified and introduced for the first time in eastern Iran. In the third season of excavations at the Kalehkoob site, the focus was on the investigation of mud-brick architectural remains that were identified in 1398 in trench E at the center of the site. Extensive excavations at this location led to the identification of architectural remains belonging to the fifth millennium BC and the Chalcolithic period. The mud-brick architecture with such antiquity is introduced for the first time. In this phase of research at the Kalehkoob site, four different architectural phases were identified.