Piliated Haemophilus influenzae type b strains display an enhanced adherence to human epithelial cells in vitro. However, clinical isolates, even from mucosal sites, are seldom piliated, although piliated populations can be selected from them. Experiments with rats have led some authors to suggest that piliation does not implement colonization by H. influenzae type b. Piliated populations were obtained from 35 strains by selection for adherence to human erythrocytes. One strain, H. influenzae H305, simultaneously acquired an increased adherence to rat erythrocytes and buccal epithelial cells. In contrast to other strains, H. influenzae H305 in piliated form was more effective than in nonpiliated form in the colonization of rats by intranasal inoculation. After the piliated inoculum, however, the colonies cultured from the nasal washes were negative for erythrocyte adherence. Thus, piliated H. influenzae type b strains have an apparent advantage to initiating colonization in the rat model but may give rise to nonpiliated progeny that are more readily cultivable from the mucosal surface.