Levels of genotypic and phenotypic diversity among 23 ampicillin-resistant, non-beta-lactamase-producing (Ampr NBLP) isolates of serologically nontypeable Haemophilus influenzae recovered from the respiratory tract were determined by multilocus enzyme electrophoresis, auxotroph testing in chemically defined media, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of penicillin-binding proteins (PBPs). Twenty distinctive multilocus enzyme genotypes were identified, among which the average level of genetic diversity per locus was equivalent to that in the species as a whole. Hence, a single, recent origin for Ampr NBLP strains is excluded. Of the growth factors tested, a requirement for methionine was significantly associated with the Ampr NBLP phenotype. In contrast to the relative homogeneity of the PBP profiles of the ampicillin-susceptible strains tested (8 PBPs detected), the PBP profiles of the Ampr NBLP strains exhibited marked heterogeneity (5 to 10 PBPs detected). Care should be taken in interpreting changes in PBP profiles and in associating these profiles with resistance for species such as H. influenzae that demonstrate variability.