A method of reverse transcription (RT) and polymerase chain reaction (PCR) for the detection of Norwalk virus in human stools was developed. A cationic detergent, cetyltrimethylammonium bromide, was found to effectively remove from stool extracts factors that inhibit the RT-PCR assay. The specificities of the tests were shown by hybridization of the amplified DNA with Norwalk virus-specific cDNA probes and a consistent correlation between virus detection in stools and infection of volunteers. RT-PCR detected virus in stool samples diluted 10(-4) and was about 100 times more sensitive than dot blot hybridization. In serial stool samples collected before and at different times after inoculation of 10 volunteers with Norwalk virus, 37 of 55 were positive by RT-PCR, but only 27 were positive by dot blot hybridization (chi 2 = 22.96; P less than 0.001). Further application of this method should allow detection of Norwalk virus in food or environmental samples such as shellfish and shellfish waters.