We have determined the nucleotide sequence of IS3. Our IS3 isolate has 39 bp inverted repeats (IR's) with 6 mismatches, and is 1258 bp long. IS3 contains a large open reading frame (ORF) of 288 codons with a smaller, partially overlapping ORF of 91 codons on the opposite strand in codon-codon register. The large ORF is preceded by and has a 4 bp overlap with a 99 codon ORF that has potential transcriptional and translational start signals. Thus, IS3 could encode a bicistronic mRNA. The Shine-Dalgarno sequence for this 99 codon ORF could be sequestered in a stem-loop structure, but only if the transcript began outside IS3, as was first seen with IS10. This could be a means for preventing fortuitous activation of IS3 by outside promoters. No DNA sequence homology was found between IS3 and other prokaryotic IS elements, but there is slight amino acid sequence homology and significant conservation of hydropathicity patterns between the putative transposases of IS3 and IS2.