A new insertion sequence designated IS1394 was isolated from Pseudomonas alcaligenes NCIB 9867 (P25X) by entrapment in plasmid pUCD800 which carries the Bacillus subtilis sacB and sacR genes. The 1100-bp sequence contains 27-bp inverted repeats with 4 bp mismatch and has one long open reading frame, spanning 92.1% of the entire IS. The deduced 338 amino-acid sequence demonstrated homology (varying from 65% to 78% similarity and 36-67% identity) to transposases encoded by the IS30 family of IS elements. Comparison of four different IS-sacB junction sequences showed that IS1394 generated 3-bp direct repeats of target DNA upon insertion. IS1394 is present in at least 10 copies in the P25X genome but none was detected in its endogenous plasmid pRA2. Hybridization experiments revealed that the distribution of IS1394 is limited to closely related strains, being present in three copies in Pseudomonas putida NCIB 9869 (P35X) and two copies in Pseudomonas alcaligenes ATCC type strain (ATCC 14904).