Lactobacillus rhamnosus GG is a human intestinal isolate that has been studied intensively because of its probiotic properties. We have previously shown that L. rhamnosus GG produces proteinaceous pili that earlier had been observed only in Gram-positive pathogens (M. Kankainen et al., Proc. Natl. Acad. Sci. U. S. A. 106:17193-17198, 2009). These pili were found to be encoded by the spaCBA gene cluster, and the pilus-associated SpaC pilin was shown to confer on the cells a mucus-binding ability. In addition to the spaCBA cluster, another putative pilus cluster, spaFED, was predicted from the L. rhamnosus GG genome sequence. Herein, we show that only SpaCBA pili are produced by L. rhamnosus, and we describe a detailed analysis of cell wall-associated and affinity-purified SpaCBA pili by Western blotting and immunogold electron microscopy. Our results indicate that SpaCBA pili are heterotrimeric protrusions with a SpaA subunit as the shaft-forming major pilin. Only a few SpaB subunits could be observed in pilus fibers. Instead, SpaB pilins were found at pilus bases, as assessed by immunogold double labeling of thin sections of cells, suggesting that SpaB is involved in the termination of pilus assembly. The SpaC adhesin was present along the whole pilus length at numbers nearly equaling those of SpaA. The relative amount and uniform distribution of SpaC within pili not only makes it possible to exert both long-distance and intimate contact with host tissue but also provides mucus-binding strength, which explains the prolonged intestinal residency times observed for L. rhamnosus GG compared to that of nonpiliated lactobacilli.