The assembly of the preinitiation complex (PIC) occurs upstream of the +1 nucleosome which, in yeast, obstructs the transcription start site and is frequently assembled with the histone variant H2A.Z. To understand the contribution of the transcription machinery in the disassembly of the +1 H2A.Z nucleosome, conditional mutants were used to block PIC assembly. A quantitative ChIP-seq approach, which allows detection of global occupancy change, was employed to measure H2A.Z occupancy. Blocking PIC assembly resulted in promoter-specific H2A.Z accumulation, indicating that the PIC is required to evict H2A.Z. By contrast, H2A.Z eviction was unaffected upon depletion of INO80, a remodeler previously reported to displace nucleosomal H2A.Z. Robust PIC-dependent H2A.Z eviction was observed at active and infrequently transcribed genes, indicating that constitutive H2A.Z turnover is a general phenomenon. Finally, sites with strong H2A.Z turnover precisely mark transcript starts, providing a new metric for identifying cryptic and alternative sites of initiation.