Nasopharyngeal carriage of Haemophilus influenzae (Hi) was studied in 114 healthy children < 3 years old, attending day-care centers (DCCs) in Ribeirão Preto, State of São Paulo, Brazil. Biotype, serotype (by specific antisera and PCR) and antibiotic susceptibility to 14 antibiotics of each isolate were determined. Carriage rates of Hi were 72.0%. Isolates belonged to biotype II (36.5%), I (21.5%), V (18.2%) and III (16.1%). The prevalence of encapsulated Hi carriers was 3.2% for type f, 1.0% for type b, 1.0% for type d and 1.0% for type e. Resistances to trimethoprim-sulphamethoxazole and ampicillin were 46.2% and 10.7% respectively. Multidrug resistance was found in 14 (15.0%) of the isolates tested. Among the isolates, 13.9% were β-lactamase producers; there were no β-lactamase negative ampicillin resistant isolates. DCCs are niches with a high potential for the spread of microorganisms and should be continuously monitored to detect elimination or replacement of H. influenzae type b colonization.