The thin pili of IncI1 plasmid R64, which is required for conjugation in liquid media, belong to the type IV pilus family. They consist of a major subunit, the pilS product, and a minor component, one of the seven pilV products. The pilS product is first synthesized as a 22-kDa prepilin, processed to a 19-kDa mature pilin by the function of the pilU product, and then secreted outside the cell. The mature pilin is assembled to form a thin pilus with the pilV product. To reveal the relationship between the structure and function of the pilS product, 27 missense mutations, three N-terminal deletions, and two C-terminal deletions were constructed by PCR and site-directed mutagenesis. The characteristics of 32 mutant pilS products were analyzed. Four pilS mutant phenotype classes were identified. The products of 10 class I mutants were not processed by prepilin peptidase; the extracellular secretion of the products of two class II mutants was inhibited; from 11 class III mutants, thin pili with reduced activities in liquid mating were formed; from 9 class IV mutants, thin pili with mating activity similar to that of the wild-type pilS gene were formed. The point mutations of the class I mutants were distributed throughout the prepilin sequence, suggesting that processing of the pilS product requires the entire prepilin sequence.