Cas9 is a bacterial RNA-guided endonuclease that uses base pairing to recognize and cleave target DNAs with complementarity to the guide RNA. The programmable sequence specificity of Cas9 has been harnessed for genome editing and gene expression control in many organisms. Here, we describe protocols for the heterologous expression and purification of recombinant Cas9 protein and for in vitro transcription of guide RNAs. We describe in vitro reconstitution of the Cas9-guide RNA ribonucleoprotein complex and its use in endonuclease activity assays. The methods outlined here enable mechanistic characterization of the RNA-guided DNA cleavage activity of Cas9 and may assist in further development of the enzyme for genetic engineering applications.