Nontypeable Haemophilus influenzae HF0295, isolated by aspiration from the middle ear of a patient with otitis media, expresses long, thick, and hemagglutinating pili of a single serotype (LKP1) on its surface. An intact pilus vaccine consisting of the LKP1 serotype protected chinchillas against experimental otitis media (C. C. Brinton, Jr., M. J. Carter, D. B. Derber, S. Kar, J. A. Kramarik, A. C. C. To, S. C. M. To, and S. W. Wood, Pediatr. Infect. Dis. J. 8:554-561, 1989; R. B. Karasic, D. J. Beste, S. C. M. To, W. J. Doyle, S. W. Wood, M. J. Carter, A. C. C. To, K. Tanpowpong, C. D. Bluestone, and C. C. Brinton, Jr., Pediatr. Infect. Dis. J. 8:562-565, 1989). The genes encoding LKP1 pili were cloned from a genomic library of the clinical strain as a 12.5-kilobase insert on a plasmid vector and inserted into Escherichia coli K-12. Transposon mutagenesis and deletion constructs mapped the pilus-coding region within a 7-kilobase region of insert DNA. The recombinant bacteria were found by electron microscopy to express pili morphologically similar to LKP1 pili. Purified pilus rods from the recombinant and its parental strain were composed of a single detectable protein with an apparent molecular weight of 27,500. Antibodies raised against LKP1 pili purified from H. influenzae immunologically reacted with pili from the recombinant bacteria. Pili from both strains also adhered to human erythrocytes and buccal cells with the same specificity.