Hepatitis A virus (HAV), a picornavirus, is the causative agent of infectious hepatitis, generally a self-limiting disease of the liver. Recently, sequences within the 5' noncoding region that affect the translation of the viral genome have been identified using in vitro systems. In this report we demonstrate that extracts prepared from mouse liver cytoplasm specifically stimulate HAV RNA translation in a rabbit reticulocyte lysate in vitro. This activity appears to act specifically on HAV sequences and is not found in other mouse tissue and several cell lines of tissue culture origin.