Rapid kinetic studies on the initiation of RNA synthesis catalyzed by Escherichia coli RNA polymerase were performed by using a multimixing stopped-flow apparatus. Tritium-labeled UTP was first mixed with a preincuBated mixture of enzyme, template poly(dA-dT), and initiator UpA to form UpAp[3H]U. After a selected time interval, the reaction mixture was mixed with a third solution containing unlabeled UTP and ATP to allow the elongation of poly(rA-rU). The net [3H]UMP incorporation into acid-precipitable product measures the formation of the initiation complex which has subsequently been elongated in RNA chains without enzyme turnover. The time course of formation of the initiation complex thus measured is biphasic with a time lag of about 30 ms and a plateau after 0.5 s. The kinetic data could be analyzed by a two-exponential equation, giving two relaxation times of about 40 and 100 ms. The presence of time lag and the apparent lack of dependence of the two relaxation times on the concentrations of UpA and [3H]UTP indicate that an inactive intermediate is formed after substrate binding to the enzyme-template complex via a rapid equilibration. This inactive intermediate is then converted into an active initiation complex. In contrast, the total relaxation amplitude shows a strong dependence of [3H]UTP concentration in a manner suggesting that more than one UTP molecule is bound to the enzyme-DNA complex during the initiation process. Based on kinetic arguments, it is proposed that the inactive intermediate is formed from the enzyme-DNA-UpA-UTP complex and converted into the enzyme-DNA-UpApU complex. Moreover, the additional UTP molecule may bind to the latter complex or the inactive intermediate mentioned above. Two minimal mechanisms of the initiation of RNA synthesis consistent with all the data are presented.