Atopy to latex has been reported in different high-risk groups of subjects in whom it is mainly caused by proteins from natural latex that are responsible for eliciting a variety of clinical symptoms, some of them systemic. Thus, identification of subjects sensitized to latex proteins is of great health importance, because it would become possible to advise them to avoid contact with latex-containing products. Protein extracts from ammoniated latex were prepared by incubation with PBS buffer either with or without detergents, followed by ultracentrifugation. Three immunoenzymic methods were developed (EAST, ELISA, and immunoblotting) to detect the presence of specific IgE antibodies against latex proteins in sera of patients from different groups at risk. The protein content of the latex extracts ranged from 5.3 to 8.8 mg/mL. The prevalence of specific IgE against latex proteins was 9/28 (32.1%) in children with multiple surgeries, 17/98 (17.3%) in health care workers, and 23/123 (18.6%) in outpatients assisted at the Allergy Department. None of the sera belonging to the healthy control group showed the presence of specific IgE. Therefore, this protein extract from latex could be used to detect specific IgE antibodies in serum by immunoenzymic methods. Serologic results for latex-specific IgE found are in accordance with those reported in the literature of other countries.