IS629 is 1,310 base pairs in length with a pair of 25-bp imperfect inverted repeats at its termini. Two partially overlapping open reading frames, orfA and orfB, are present in IS629, and two putative translational frameshift signals, TTTTG (T₄G) and AAAAT (A₄T), are located near the 3’ end of orfA. With the lacZ gene as the reporter, both T₄G and A₄T motifs are determined to be a -1 frameshift signal. Two peptides representing the two transframe products designated OrfAB’ and OrfAB, are identified by a liquid chromatography-tandem mass spectrometric (LC-MS/MS) approach. Results of transposition assays show that OrfAB’ is the transposase and that OrfAB aids in the transposition of IS629. Pulse-chase experiments and E. coli two-hybrid assays demonstrate that OrfAB binds to and stabilizes OrfAB’, thus increasing the transposition activity of IS629. This is the first transposable element in the IS3 family shown to have two functional frameshifted products involved in transposition and to use a transframe product to regulate transposition.