Restriction-modification controller proteins play an essential role in regulating the temporal expression of restriction-modification genes. The controller protein C.Csp231I represents a new class of controller proteins. The gene was sublconed to allow overexpression in Escherichia coli. The protein was purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.0 A resolution and belonged to space group P2(1). An electrophoretic mobility-shift assay provided evidence of strong binding of C.Csp231I to a sequence located upstream of the csp231IC start codon.