Runting stunting syndrome (RSS) is an enteric disease of young broiler chickens characterized by weight suppression and poor flock uniformity. Cystic lesions in the crypt of Lieberkhn in the small intestines are typical for RSS. Despite indications of viral etiology, the etiologic agent(s) were unknown. Based on recently described vaccination experiments, a novel chicken astrovirus (CkAstV) was suggested as an RSS pathogen. The aim of these studies was to investigate the etiological role of the novel CkAstV in RSS. Three different astroviruses (ANV-1, ANV-2, new CkAstV) were detected in the intestines of RSS-affected chickens by in situ hybridization. In subsequent studies, only the CkAstV-specific signals were detected in the cystic lesions, implying a close association with the formation of the RSS hallmark lesion. Furthermore, the full length nucleotide sequence of the viral genome of a CkAstV present in gut homogenates obtained from RSS-affected chickens was determined. The sequence showed significant differences compared to published sequences of other chicken astroviruses. Additionally, a different replication mechanism for this virus was postulated based on differences for the initiation of expression of the viral RNA-dependent RNA polymerase. In experiments performed in parallel, a new CkAstV was isolated in cell culture. This new CkAstV (cCkAstV) replicated in chickens in the intestinal crypt epithelial cells during the first few days of life. However, during the first passage in chickens, the cCkAstV did not induce typical clinical signs of RSS. A serial chicken-to-chicken passage of the cCkAstV resulted in an increased virulence during the 4th passage and showed all signs of RSS. Comparison of the complete genomic sequence of the cCkAstV with the chicken passaged cCkAstV revealed only minor genetic modification as indicated by a >99% homology which resulted in a total of four amino acid exchanges in two viral proteins. The consequences of the observed mutations need to be further evaluated utilizing a reverse genetics approach. In conclusion, the results indicated that the cCkAstV has the potential to cause clinical signs of RSS in chickens. As an etiological agent of the disease, the involvement of the cCkAstV in RSS was strongly supported by this study.