To address the relationship between chromatin structure and histone gene expression, the nucleosomal organization of a cell cycle-dependent human H4 histone gene in a bovine papilloma virus (BPV) minichromosome was examined. The nucleosome repeat length of the human H4 histone gene, maintained as a stable episome in a C127 mouse cell line designated I-8, was compared with that of the chromosomal copy of the H4 gene in human (HeLa) cells. In both cell lines, the H4 histone gene is predominantly expressed during the S phase of the cell cycle. The nucleosome repeat length of total HeLa cell and C127 mouse cell chromatin was similarly examined. Nuclei were digested with micrococcal nuclease and the DNA was fractionated electrophoretically, transferred to nitrocellulose filters and hybridized with radiolabelled (32P) cloned DNA probes. The nucleosome repeat length of the H4 gene, as an episome in the C127 mouse cell (153 +/- 8) and as an integrated copy in a HeLa cell (163 +/- 10) was considerably shorter than total genomic host cell (C127) (190 +/- 5) or HeLa cell chromatin (183 +/- 7). Our results indicate that the episomal H4 histone gene is packaged as chromatin. Moreover, the shortened nucleosome repeat length of the H4 gene, both as an episome or integrated chromosome sequence, suggests that the repeat length is characteristic of the gene and may be functionally related to its cell cycle regulated expression.