A kinetic characterization is presented for intron insertion into a target duplex DNA site during L1.ltrB intron mobility. This reaction is catalyzed by a ribonucleoprotein particle (RNP), consisting of a lariat form of group II intron RNA and the intron-encoded LtrA protein. In the first stage of intron mobility, the RNA component of the enzyme by itself inserts directly into the target ds DNA site, and thus, the RNP enzyme does not carry out turnover. Using single-turnover kinetics, we established an in vitro kinetic assay system and investigated mechanism of the intron RNA insertion process.