Ampicillin resistance in Haemophilus influenzae and Neisseria gonorrhoeae is most commonly due to plasmid-mediated production of the TEM β-lactamase. The H injluenzae plasmids may have evolved by insertion of various antibiotic resistance transposons into a phenotypically cryptic plasmid found in one of 699 isolates of H. influenzae examined. The small, nonconjugative, β-lactamase-specifying plasmids of N. gonorrhoeae and Haemophilus species are highly related. Phenotypically cryptic plasm ids found in several epidemiologically distinct isolates of Haemophilus parainfluenzae are highly related to the β-lactamase plasmids but carry no transposon A (TnA) sequences. This evidence strongly favors the hypothesis that the β-lactamase plasmids evolved by the insertion of TnA (possibly introduced from enteric bacteria) into cryptic plasmids resident in H. parainjluenzae.