Previous studies have identified several biochemical parameters that resolve promoter-specific transcription initiation by RNA polymerase II into distinct steps. We have employed these parameters to compare the mechanisms of initiation mediated through either a TATA box or a transcriptional initiator element. Time course experiments revealed that initiator-mediated transcription in a cell-free extract does not display the lag period typically found with TATA-mediated transcription. Titration experiments with increasing amounts of extract revealed further differences between TATA- and initiator-mediated transcription, again suggesting that different steps are rate-limiting. In contrast, experiments with the detergent Sarkosyl, which is believed to define later steps in the initiation process, revealed a striking degree of similarity between TATA- and initiator-mediated transcription. Additional similarities were found using dinucleotide priming experiments, which are believed to measure the flexibility of initiation after RNA polymerase II is positioned over the transcription start site region. Taken together, these results suggest that the location of a preinitiation complex can be determined by core promoter recognition through either a TATA box or an initiator element. However, following these distinct template recognition events, the steps leading to initiation may be very similar and may proceed in a DNA sequence-independent manner.