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To determine the prevalence and risk factors for Toxoplasma gondii infection in Guatemalan children, in 1999 and 2003 we surveyed caretakers and serologically tested children in the San Juan Sacatepequez area using Platelia Toxo IgG TMB enzyme immunoassay kits. In 1999, of 532 children six months to two years old, 66 (12.4%) were antibody positive. In 2003, in 500 children 3–10 years old antibody prevalence increased from 24% to 43% at age five years then leveled off. By multivariate analysis, drinking well water (relative risk [RR] = 1.78, 95% confidence limit [CL] = 1.00, 3.17, P = 0.05) and not cleaning up cat feces (RR = 2.06, 95% CL = 1.00, 4.28, P = 0.05) increased the risk of T. gondii seropositivity. Most T. gondii infections in children from these villages occurred by age five, but half were still not infected by adolescence. Therefore, it is important to educate girls entering child-bearing age about the risks of acute T. gondii infection and the local risk factors for infection.