The CRISPR-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA:DNA base-pairing to target foreign DNA in bacteria. This study investigates how Cas9:RNA complexes interrogate DNA to find specific cleavage sites. Single-molecule and bulk biochemical experiments reveal that both binding and cleavage of DNA by Cas9:RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9:RNA. DNA strand separation and RNA:DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. PAM interactions trigger Cas9 catalytic activity. These findings explain how Cas9 employs PAM recognition to quickly identify potential target sites while scanning large DNA molecules and to regulate dsDNA scission.The CRISPR-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA:DNA base-pairing to target foreign DNA in bacteria. This study investigates how Cas9:RNA complexes interrogate DNA to find specific cleavage sites. Single-molecule and bulk biochemical experiments reveal that both binding and cleavage of DNA by Cas9:RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9:RNA. DNA strand separation and RNA:DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. PAM interactions trigger Cas9 catalytic activity. These findings explain how Cas9 employs PAM recognition to quickly identify potential target sites while scanning large DNA molecules and to regulate dsDNA scission.