Although the pili of Gram-positive bacteria are putative virulence factors, little is known about their structure. Here we describe the molecular architecture of pilus-1 of Streptococcus pneumoniae, which is a major cause of morbidity and mortality worldwide. One major (RrgB) and two minor components (RrgA and RrgC) assemble into the pilus. Results from TEM and scanning transmission EM show that the native pili are approximately 6 nm wide, flexible filaments that can be over 1 microm long. They are formed by a single string of RrgB monomers and have a polarity defined by nose-like protrusions. These protrusions correlate to the shape of monomeric RrgB-His, which like RrgA-His and RrgC-His has an elongated, multi-domain structure. RrgA and RrgC are only present at the opposite ends of the pilus shaft, compatible with their putative roles as adhesin and anchor to the cell wall surface, respectively. Our structural analyses provide the first direct experimental evidence that the native S. pneumoniae pilus shaft is composed exclusively of covalently linked monomeric RrgB subunits oriented head-to-tail.