Water and sanitation interventions in developing countries have historically been difficult to evaluate. We conducted a seroepidemiologic study with the following goals: 1) to determine the feasibility of using antibody markers as indicators of waterborne pathogen infection in the evaluation of water and sanitation intervention projects; 2) to characterize the epidemiology of waterborne diarrheal infections in rural Guatemala, and 3) to measure the age-specific prevalence of antibodies to waterborne pathogens. Between September and December 1999, all children 6-36 months of age in 10 study villages were invited to participate. We collected sufficient serum from 522 of 590 eligible children, and divided them into six-month age groups for analysis (6-12, 13-18, 19-24, 25-30, and 31-36 months). The prevalence of antibodies was lowest in children 6-12 months old compared with the four older age groups for the following pathogens: enterotoxigenic Escherichia coli (48%, 81%, 80%, 77%, and 83%), Norwalk virus (27%, 61%, 83%, 94%, and 94%), and Cryptosporidium parvum (27%, 53%, 70%, 67%, and 73%). The prevalence of total antibody to hepatitis A virus increased steadily in the three oldest age groups (40%, 28%, 46%, 60%, and 76%). In contrast, the prevalence of antibody to Helicobacter pylori was relatively constant in all five age groups (20%, 19%, 21%, 25%, and 25%). Serology appears to be an efficient and feasible approach for determining the prevalence of infection with selected waterborne pathogens in very young children. Such an approach may provide a suitable, sensitive, and economical alternative to the cumbersome stool collection methods that have previously been used for evaluation of water and sanitation projects.