Utilizing yeast strains containing promoter mutations, we demonstrate that transcription of the HSP82 gene causes nucleosomes toward the 3'-end to become DNase I sensitive and 'split' into structures that exhibit a 'half-nucleosomal' cleavage periodicity. Splitting occurs even when only a few RNA polymerase II molecules are engaged in basal level transcription or during the first round of induced transcription. The split nucleosomal structure survives nuclear isolation suggesting that it may be stabilized by post-translational modifications or non-histone proteins, and may require DNA replication for reversal to a whole nucleosomal structure. Split nucleosomes represent a structure for DNase I sensitive chromatin and are probably of common occurrence but difficult to detect experimentally. We suggest that transient positive supercoils downstream of traversing RNA polymerase lead to nucleosome splitting.