Eighty-seven serum specimens from 20 human subjects experimentally inoculated one or more times with Norwalk virus were quantitatively examined for virus-specific immunoglobulin M (IgM). A sensitive and specific radioimmunoassay for anti-Norwalk virus blocking activity was applied to whole serum and to separate IgM and IgG fractions obtained by sucrose density gradient ultracentrifugation. The peak IgM response occurred at about 2 weeks after illness, but IgM was detectable at lower titers for up to 21 weeks after infection. The IgM response was seen in volunteers who became ill, whether or not prechallenge total serum antibody was present. On long-term (27 to 42 months) rechallenge, volunteers who were previously ill and had produced IgM antibody again developed illness, and a secondary IgM response greater than the first was detected. Inoculated volunteers who did not develop illness, as well as previously ill volunteers on short-term rechallenge (4 to 14 weeks), usually failed to generate an IgM response, whether or not an IgG response had occurred. In ill subjects, the rise in IgM and IgG occurred concomitantly. Virus-specific IgM is not necessarily indicative of primary infection with Norwalk agent inasmuch as reinfection produces an enhancement of the IgM response. Furthermore, Norwalk-specific IgM responses do not appear to be associated with subclinical illness.