Antimicrobial resistance determinants and plasmids present in 10 multiply antibiotic-resistant strains of Hemophilus influenzae isolated from patients in different geographic regions of Spain were characterized. Conjugative plasmids with molecular sizes of 38-50 MDa encoded resistance to ampicillin, chloramphenicol, kanamycin, streptomycin, sulfamethoxazole, and tetracycline. Trimethoprim resistance was not linked to the other antibiotic resistance determinants and trimethoprim-resistant transconjugants and transformants lacked detectable plasmid DNA, suggesting that this determinant is chromosomal. Restriction endonuclease analysis revealed similarities among the plasmids, but several restriction patterns could be distinguished. Three hybridization patterns were found with DNA probes coding for H. parainfluenzae beta-lactamase and chloramphenicol-acetyltransferase. Resistance to kanamycin was due to drug modification by aminoglycoside-phosphotransferase (3')I. In Spain, it appears that multiple antibiotic resistance phenotypes in H. influenzae did not arise from acquisition of a single R plasmid; rather, both plasmid and chromosomal resistance evolved independently from several sources.