Hepatitis A virus (HAV), a major cause of infectious hepatitis in humans, is a positive strand RNA virus belonging to the hepatovirus group of the picornavirus family. Primary detection of HAV in clinical or biological samples is not routinely possible at present because wild-type HAV grows very poorly in cell culture. Except for virus preparations that have been adapted for rapid growth in cell culture, HAV does not produce a detectable cytopathic effect in infected cells. For these reasons, several laboratories have attempted to develop methods based on nucleic acid hybridization or the polymerase chain reaction (PCR) for detecting HAV. The reverse transcription-PCR (RT-PCR) procedure described here for detecting HAV in shellfish can be adapted for use in clinical samples, which have a much higher viral load and which contain less extraneous material that could interfere with RT-PCR. Also described here are the synthesis and use of a competitor template RNA for determination of the number of HAV genomic RNA molecules in a sample. We emphasize, however, that the method described here does not provide information about the infectious viral load in a sample, but simply the number of RNA molecules having the HAV specific sequence.