We examined nine chloramphenicol-resistant (minimal inhibitory concentration, greater than or equal to 15 micrograms/ml) Haemophilus influenzae strains isolated in various parts of the world to characterize the genetic and biochemical bases of the resistance; four were type b. All nine contained conjugative plasmids, ranging in molecular weight from 34 x 10(6) to 46 x 10(6), which encoded for resistance to chloramphenicol and tetracycline or chloramphenicol, tetracycline, and ampicillin. Deoxyribonucleic acid homology studies showed that these plasmids were closely related to a previously described ampicillin-resistant plasmid, RSF007, and to each other. All nine isolates and their chloramphenicol-resistant transconjugants produced chloramphenicol acetyltransferase. We conclude that chloramphenicol resistance in these strains of H. influenzae is via plasmid-mediated production of chloramphenicol acetyltransferase.