The expression within Pseudomonas aeruginosa PAO1 of three mutant pilin genes from P. aeruginosa PAK was studied to determine their effects on pilin stability, translocation into the membrane, leader peptide removal, and methylation of the mature N-terminal phenylalanine. The results revealed that a deletion of 4 or 8 amino acids within the immediate N-terminus of pilin had deleterious effects upon leader peptide cleavage. In addition, while the 4-amino-acid deletion did not affect pilin partitioning into the membrane, the 8-amino-acid deletion decreased the amount of pilin found within the membrane fraction. Of considerable interest was the finding that the mutation within the mature pilin of the glutamate at position 5 to a lysine did not prevent leader peptide removal but did inhibit the methylation of the N-terminal phenylalanine.