Bacteriophage T7 gp4A' protein is a hexameric helicase-primase protein that separates the strands of a duplex DNA in a reaction coupled to dTTP hydrolysis. Here we reexamine in more detail the kinetic mechanism of dTTP hydrolysis by a preassembled T7 helicase hexamer in the absence of DNA. Pre-steady state dTTP hydrolysis kinetics showed a distinct burst, whose amplitude indicated that a preformed hexamer of T7 helicase hydrolyzes on an average one dTTP per hexamer. The pre-steady state chase-time experiments provided evidence for sequential hydrolysis of two dTTPs. The medium [18O]Pi exchange experiments failed to detect dTTP synthesis indicating that the less than six site hydrolysis observed is not due to reversible dTTP hydrolysis on the helicase active site. The Pi-release rate was measured directly using a stopped-flow fluorescence assay and it was found that the rate of dTTP hydrolysis on the helicase active site is 8 times faster than the Pi-release rate, which in turn is 3 times faster than the dTDP release rate. Thus, the rate-limiting step in the pathway of helicase-catalyzed dTTPase reaction is the release of dTDP. Chase-time dTTPase kinetics in the steady state phase provided evidence for two to three slowly hydrolyzing dTTPase sites on the hexamer. The results of this study are therefore consistent with those reported earlier (Hingorani, M. M., Washington, M. T., Moore, K. C., and Patel, S. S. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 5012-5017) and they support a model of dTTP hydrolysis by T7 helicase hexamer that is similar to the binding change mechanism of F1-ATPase with dTTP hydrolysis occurring sequentially at the catalytic sites.