The transposase family of proteins mediate DNA transposition or retroviral DNA integration via multistep phosphoryl transfer reactions. For Tn10 and phage Mu, a single active site of one transposase protomer catalyzes the successive transposition reaction steps. We examined phosphorothioate stereoselectivity at the scissile position for all four reaction steps catalyzed by the Tn10 transposase. The results suggest that the first three steps required for double-strand cutting at the transposon end proceed as a succession of pseudo-reverse reaction steps while the 3' end of the transposon remains bound to the same side of the active site. However, the mode of substrate binding to the active site changes for the cut transposon 3' end to target DNA strand joining. The phosphorothioate stereoselectivity of the corresponding steps of phage Mu transposition and HIV DNA integration matches that of Tn10 reaction, indicating a common mode of substrate-active site interactions for this class of DNA transposition reactions.