Histone variant H2A.Z-containing nucleosomes are incorporated at most eukaryotic 
promoters. This incorporation is mediated by the conserved SWR1 complex, which 
replaces histone H2A in canonical nucleosomes with H2A.Z in an ATP-dependent 
manner. Here, we show that promoter-proximal nucleosomes are highly 
heterogeneous for H2A.Z in Saccharomyces cerevisiae, with substantial 
representation of nucleosomes containing one, two, or zero H2A.Z molecules. 
SWR1-catalyzed H2A.Z replacement in vitro occurs in a stepwise and 
unidirectional fashion, one H2A.Z-H2B dimer at a time, producing heterotypic 
nucleosomes as intermediates and homotypic H2A.Z nucleosomes as end products. 
The ATPase activity of SWR1 is specifically stimulated by H2A-containing 
nucleosomes without ensuing histone H2A eviction. Remarkably, further addition 
of free H2A.Z-H2B dimer leads to hyperstimulation of ATPase activity, eviction 
of nucleosomal H2A-H2B, and deposition of H2A.Z-H2B. These results suggest that 
the combination of H2A-containing nucleosome and free H2A.Z-H2B dimer acting as 
both effector and substrate for SWR1 governs the specificity and outcome of the 
replacement reaction.