The accessibility of nucleosomal DNA to transcription factors and other sequence-specific DNA binding proteins is of importance in the consideration of mechanisms of transcriptional control. Here, we report a simple novel assay which determines this accessibility at eight different rotationally equivalent sites on nucleosomal DNA and shows that linker histones and the chromosomal HMGB proteins, HMG-D and HMG-Z, have opposite effects on the accessibility of nucleosomal DNA. We compare this assay to previously described methods.