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ABSTRACTHelicobacter pyloricauses numerous alterations in gastric epithelial cells through processes that are dependent on activity of the cagtype IV secretion system (T4SS). Filamentous structures termed “pili” have been visualized at the interface between H. pyloriand gastric epithelial cells, and previous studies suggested that pilus formation is dependent on the presence of the cagpathogenicity island (PAI). Thus far, there has been relatively little effort to identify specific genes that are required for pilus formation, and the role of pili in T4SS function is unclear. In this study, we selected 7 genes in the cagPAI that are known to be required for T4SS function and investigated whether these genes were required for pilus formation. cagT, cagX, cagV, cagM, and cag3mutants were defective in both T4SS function and pilus formation; complemented mutants regained T4SS function and the capacity for pilus formation. cagYand cagCmutants were defective in T4SS function but retained the capacity for pilus formation. These results define a set of cagPAI genes that are required for both pilus biogenesis and T4SS function and reveal that these processes can be uncoupled in specific mutant strains.