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The route of transmission of Helicobacter pylori (H. pylori) is unclear. Gastro-oral transmission via contaminated vomitus has been proposed as an important mode of transmitting H. pylori, especially in children. This pilot study attempted to isolate H. pylori from the vomitus of children.Children presenting for evaluation with gastroenteritis-associated vomiting were studied. Fresh vomitus samples were collected for detection of H. pylori by bacteriological culture and polymerase chain reaction (PCR). A rapid, whole blood test was used to determine the H. pylori status of patients.A total of 18 children with mean age of 6 yr were studied; four had a positive serology test. Among these four children, H. pylori was isolated from vomitus by culture in one child and by PCR in two. An 18-month-old girl with negative serology had H. pylori detected in vomitus by PCR. Six months later, she had seroconversion confirmed, suggesting that she had an acute H. pylori infection on initial presentation.This is the first study reporting successful isolation of H. pylori from naturally produced vomitus. The result implies that transmission of H. pylori infection by vomitus, especially in children, is possible.