Analysis of spontaneous mutations in Acinetobacter calcoaceticus revealed a 1237 bp insertion sequence named IS7236 and possessing a nucleotide sequence resembling those of members of the IS3 family. The chromosome of A. calcoaceticus strain ADPl contains seven copies of IS7236 which appears to insert preferentially into pobR, the transcriptional activator of the structural gene for p-hydroxybenzoate hydroxylase. IS7236 creates tandem 3 bp DNA duplications flanking the sites of its insertion in pobR. Different duplication patterns are found following insertion of IS7236 into pcaH, a structural gene for protocatechuate 3,bdioxygenase. Therefore the insertion properties of IS7236 appear to be influenced by its DNA target. Amino acid sequences associated with the apparent transposase function have been conserved in ORFB of IS7236 whereas the presumed DNA-binding helix-turn-helix region of IS7236 ORFA exhibits substantial amino acid sequence divergence from its IS3 counterparts. IS7236 ORFA and ORFB coding sequences overlap considerably, and sequence evidence indicates mechanisms for ORFB expression in IS7236 may resemble those employed by other members of the IS3 family. Portions of the IS7236 terminal repeats exhibit substantial sequence divergence from other members of the IS3 family, but evolution appears to have conserved a mechanism preventing expression of the insertion sequence genes as a consequence of transcriptional readthrough.