Small round-structured viruses (SRSVs) are a genetically and antigenically diverse group of caliciviruses that are the most common cause of outbreaks of acute nonbacterial gastroenteritis. We have applied both molecular techniques to characterize SRSVs in fecal specimens and serologic assays using four different expressed SRSV antigens to examine the distribution of outbreak strains in the United States and determine if the immune responses of patients were strain specific. Strains from 23 outbreaks of SRSV gastroenteritis were characterized by reverse transcription-PCR and nucleotide sequencing of a 277-base region of the capsid gene. These strains segregated into two distinct genogroups, I and II, comprising four and six clusters of strains respectively, each representing a distinct phylogenetic lineage. Serum IgG responses in patients were measured by enzyme immunoassay using expressed capsid antigens of Norwalk virus (NV), Toronto virus (TV), Hawaii virus (HV), and Lordsdale virus (LV), representing four of the 10 clusters. While strains in genogroups I and II were antigenically distinct, within genogroups, the specificity of the immune response varied greatly. Patients infected with genogroup I strains which had as much as 38.5% aa divergence from NV demonstrated relatively homologous seroresponses to the single NV antigen. In contrast, in genogroup II, homologous seroresponses to TV and HV were only present when the infecting strains showed less than 6.5% aa divergence from these antigens. These results suggest that TV and HV represent not only separate genetic clusters in genogroup II but also separate antigenic groups, each of which is related but distinguishable. In addition, two genetically distinct SRSV strains were identified for which we have no homologous antigen. This study suggests that while current molecular diagnostics are capable of detecting the full range of SRSVs, additional expressed antigens will be required to detect an immune response to SRSV infection caused by all the antigenically diverse strains.