The absolute temporal couplings between DNA binding and base flipping were examined for the EcoRI DNA methyltransferase. The binding event (monitored using rhodamine-x fluorescence anisotropy) was monophasic with a second-order on-rate of 1.1 x 10(7) M-1 s-1 </= kon </= 2.25 x 10(7) M-1 s-1. Base-flipping kinetics (monitored using 2-aminopurine fluorescence intensity) were essentially synchronous with the binding kinetics, with less than a 4 ms delay between enzyme binding and target base flipping. The 4 ms delay translates into a base-flipping rate of at least 195 s-1, when the data are analyzed in terms of a sequential DNA binding and base-flipping reaction mechanism. Synchrony of binding and base flipping was only observed during the first 80% of the reaction, and an additional 20% base-flipping signal occurred well after DNA binding was complete. This additional 2AP fluorescence change, with an effective rate of 0.55 s-1, is an intramolecular isomerization reaction which greatly accelerates the dissociation of the enzyme from DNA. The correlation between the dissociation of the enzyme-DNA complex and the restacking of the extrahelical base also revealed a very tight coupling of these two events. Both dissociation and base restacking were found to be biphasic. These data are consistent with the following mechanism. The initial binding rate and base-flipping rates map very closely with previously determined pre-steady-state burst-rate kinetics for methyl transfer. Hence, binding, flipping, and methylation appear to occur in nearly a single concerted step. The bound complex then slowly isomerizes (0.1 s-1) to a distinct configuration that accelerates the product-release phase of the reaction. The product-release enzyme configuration dissociates from DNA approximately 8 times faster than the initial bound complex (0.18 s-1 vs 0.024 s-1 ). When the enzyme dissociates from the DNA along the product-release pathway, the target base remains in an extrahelical conformation and restacks at a rate of only 0.6 s-1. This "multicolor" fluorescence kinetic approach directly measures the absolute temporal correlation between DNA binding and base flipping, with millisecond timing resolution. The data reveal that even when the B-DNA structure is altered in a radical manner (e.g., via base flipping), enzymes can perform this operation in a highly efficient, if not completely concerted manner.