Escherichia coli K-12 possesses at least 16 chromosomal genes related to genes involved in the formation of type IV pili in other gram-negative bacteria. However, E. coli K-12 does not produce type IV pili when grown under standard laboratory conditions. The results of reverse transcription-PCR, operon fusion analysis, and immunoblotting demonstrated that several of the putativeE. coli piliation genes are expressed at very low levels. Increasing the level of expression of the major pilin gene (ppdD) and the linked assembly genes hofB andhofC (homologues of the Pseudomonas aeruginosatype IV pilus assembly genes pilB and pilC) did not lead to pilus production. However, expression of theppdD gene in P. aeruginosa led to assembly of PpdD into pili that were recognized by antibodies directed against the PpdD protein. Assembly of PpdD into pili in P. aeruginosawas dependent on the expression of the pilB andpilC genes and independent of expression of the P. aeruginosa pilin structural gene pilA.