Of 2458 isolates of Haemophilus influenzae examined in a recent British survey, 42 were resistant to chloramphenicol. Two resistant isolates were of type b and 40 were non-capsulate. Spectrophotometric assay showed that all the resistant isolates produced chloramphenicol acetyltransferase (CAT). CAT activity did not increase following growth on heated blood agar containing chloramphenicol 2 mg/L but was reduced by 84-98% when extracts were treated for 30 min with 5', 5'-dithiobis-2-nitrobenzoate. These data suggest that H. influenzae CATs resemble the Type-II CATs produced by enterobacteria. Extrachromosomal DNA was detected in five only of the 42 resistant isolates and cured derivatives of two plasmid-containing strains retained their chloramphenicol resistance. These results suggest that the CAT gene is located on the chromosome.