Enterococcus (Streptococcus) faecalis transposon Tn916 was introduced into Haemophilus influenzae Rd and Haemophilus parainfluenzae by transformation and demonstrated to transpose efficiently. Haemophilus transformants resistant to tetracycline were observed at a frequency of approximately 3 x 10(2) to 5 x 10(3)/micrograms of either pAM120 (pGL101::Tn916) or pAM180 (pAM81::Tn916) plasmid DNAs, which are incapable of autonomous replication in this host. Restriction enzyme analysis and Southern blot hybridization revealed that (i) Tn916 integrates into many different sites in the H. influenzae and H. parainfluenzae genomes; (ii) only the 16.4-kilobase-pair Tn916 DNA integrates, and no vector DNA was detected; and (iii) the Tetr phenotype was stable in the absence of selective pressure. Second-generation Tn916 transformants occurred at the high frequency of chromosomal markers and retained their original chromosomal locations. Similar results were obtained with H. influenzae Rd BC200 rec-1 as the recipient strain, which suggests host rec functions are not required in Tn916 integrative transposition. Transposition with Tn916 is an important procedure for mutagenesis of Haemophilus species.