The O antigen of the Helicobacter pylori lipopolysaccharide is composed of repeating units of fucosylated Lewis (Le) antigens. The α(1,2)-fucosyltransferase ( futC) of H. pylori, which catalyzes the conversion of Le x to Le y by addition of fucose, is subject to slipped-strand mispairing involving a homonucleotide (poly-C) tract. To explore the distribution of Le phenotypes within H. pylori cells grown in vitro, 379 single colonies of strain J166 were examined for Le expression. Two major populations with reciprocal Le x/Le y phenotypes were identified. Phenotypes correlated with futC frame status, suggesting that strain J166 represents a mixed population with respect to futC poly-C tract length, which was confirmed by a translational reporter. After hundreds of generations in vitro, phenotypes did not change significantly, indicating that the observed J166 Le diversity reflects the founding population. Since slipped-strand mispairing in the futC poly-C tract was postulated to explain the Le y phenotypic change observed in J166 derivative strain 98–169 isolated 10 months after rhesus monkey challenge, in trans complementation with in-frame futC was performed. Le y synthesis was restored and Le x expression was reciprocally lowered. From these studies, we confirmed the principal role of futC slipped-strand mispairing in Le antigenic variation in vitro and in vivo.