Site-directed mutagenesis was employed to create lesions in fimI, a gene of uncertain function located in the chromosomal gene cluster (fim) involved in Escherichia coli type 1 pilus biosynthesis. Chromosomal fimI mutations produced a piliation-negative phenotype. Complementation analysis indicated that a fimI'-kan insertion mutation and a fimI frameshift mutation produced polarity-like effects not seen with an in-frame fimI deletion mutation. Minicell analysis associated fimI with a 16.4-kDa noncytoplasmic protein product (FimI). We conclude that FimI has a required role in normal pilus biosynthesis.