Small round-structured viruses (SRSVs) or Norwalk-like viruses have been implicated as an important causative agent of gastroenteritis outbreaks. We used the relatively newly developed recombinant enzyme immunoassays (EIAs) to determine the seroprevalence of Norwalk virus (NV) and Mexico virus (MxV) in a family-based cohort and an antenatal clinic cohort at Ga-Rankuwa Hospital, Ga-Rankuwa, South Africa. High prevalences (96 to 99%) of anti-NV and anti-MxV antibodies were detected in both cohorts. We also investigated the pattern of antibody acquisition in a cohort of infants and young children without gastroenteritis and found that by 48 months of age all children had acquired adult antibody levels to both these viruses. Lastly, we tested 276 stool specimens collected from infants and young children with gastroenteritis for the presence of NV or MxV antigen by recombinant EIAs to each virus, by electron microscopy (EM), and by reverse transcription (RT)-PCR. NV and MxV antigens were present in 1.8 and 4.3% of the stool specimens, respectively, by the recombinant EIAs; 9.2% were positive for SRSVs by EM, and 25% of these SRSVs gave a positive result by RT-PCR for primer pair 35-36 directed to a region of the RNA-dependent RNA polymerase gene. The seroprevalence studies indicate a high level of exposure to these viruses in both children and adults. Although the viral antigens are not highly prevalent in diarrheal stools, it was determined by the two assays for NV and MxV that children are, nevertheless, infected early in life.