The Hawaii agent is a Norwalk-like virus of acute gastroenteritis in humans which is antigenically distinct from the prototype Norwalk agent. We established a solid phase sandwich type microtiter enzyme immunoassay (EIA) for Hawaii antigen employing sera and stools from experimentally challenged volunteers as reagents. This assay detected the Hawaii agent in stools from 3 of 8 volunteers who were ill after oral challenge with the Hawaii agent, including one specimen which was positive to a dilution of 1/320. Virus shedding occurred on days 3 to 7 after challenge. The Hawaii-positive stools did not react in the EIAs for Norwalk and Snow Mountain agent (SMA), nor did Norwalk or SMA-positive stools react in the Hawaii EIA. Human rotavirus, enteric adenovirus, feline calicivirus, and several enteroviruses also did not react in the Hawaii EIA. A blocking EIA to detect serum antibody to the Hawaii agent was also developed employing a diarrheal stool containing Hawaii as a source of antigen. Serum antibody rises were detected in 15 of 16 individuals with experimentally induced illness after challenge and in 3 of 5 individuals who remained well after challenge. The EIA for the Hawaii agent should permit epidemiologic studies of the Hawaii agent to be carried out as well as allow further characterization of the Hawaii virion.