The Saccharomyces cerevisiae origin recognition complex (ORC) is bound to origins of DNA replication throughout the cell cycle and directs the assembly of higher-order protein-DNA complexes during G(1). To examine the fate of ORC when origin DNA is unwound during replication initiation, we determined the effect of single-stranded DNA (ssDNA) on ORC. We show that ORC can bind ssDNA and that ORC bound to ssDNA is distinct from that bound to double-stranded origin DNA. ssDNA stimulated ORC ATPase activity, whereas double-stranded origin DNA inhibited the same activity. Electron microscopy studies revealed two alternative conformations of ORC: an extended conformation stabilized by origin DNA and a bent conformation stabilized by ssDNA. Therefore, ORC appears to exist in two distinct states with respect to its conformation and ATPase activity. Interestingly, the effect of ssDNA on these properties of ORC is correlated with ssDNA length. Since double-stranded origin DNA and ssDNA differentially stabilize these two forms of ORC, we propose that origin unwinding triggers a transition between these alternative states.