The role of pili of Haemophilus influenzae type b (Hib) in binding to human erythrocytes and in colonization and invasion of human nasopharyngeal (NP) organ cultures has been evaluated. Hib strains 1009 and 1007, NP and cerebrospinal fluid isolates from the same child with Hib meningitis, were studied. Strain 1009 was piliated (P+), produced pilin of approximately 24 kDa, and was hemadsorption-positive (HA+); strain 1007 was nonpiliated (P-), did not produce pilin, and was hemadsorption-negative (HA-). The rate of transition from one hemadsorption phenotype to the other in broth cultures and NP organ culture supernatants was 3 x 10(-4) per bacterium per generation for HA+ to HA- and 7 x 10(-4) per bacterium per generation for HA- to HA+. Growth in human NP organ culture supernatants of the P+HA+ strain was greater than that of the P-HA- strain at 6 and 12 h after infection. No difference was noted when the strains were grown in nutrient broth. Strain 1009 (P+HA+) attached in large clusters to cellular debris and nonciliated cells, a phenomenon never noted with strain 1007 (P-HA-). NP organ cultures infected with strain 1007 showed greater mucosal invasion than those infected with the 1009 strain. While P+HA+ and P-HA- Hib both attached to NP mucosa, P+HA+ strains may have a selective advantage in mucosal colonization but P-HA- strains may be more invasive.