To approach experimentally changes of chromatin structure introduced by glucocorticoids, the histone H1 compositions of hormone-treated and non-treated mouse mammary tumor cells of the GR line [Ringold, G., Lasfargues, E. Y., Bishop, J. M. and Varmus, H. E. (1975) Virology 65, 135-147] were compared. To define the biologically important hormone concentration range, the cells were exposed to different concentrations of triamcinolone, a synthetic glucocorticoid. The induction of mouse mammary tumor virus (MMTV) RNA was measured by cDNA excess hybridization, and the amount of hormone bound to nuclei was determined by a filter-binding assay. Between 0.3 nM and 30 nM triamcinolone the relative increase in nuclear bound hormone corresponded well with the relative induction of MMTV RNA. The half-life of triamcinolone in nuclei of growing cells was 1 h, as measured by a pulse-chase experiment. Reversed-phase high-performance liquid chromatography of histone H1 resulted in its separation into four subfractions. The treatment of cells with biologically active glucocorticoid, 3 nM or 30 nM triamcinolone or 1 microM dexamethasone, resulted in changes in the relative amounts of two subfractions and to a positional shift of two subfractions as compared to untreated cells. No changes were observed after exposure to 3 nM dexamethasone, a concentration which does not induce MMTV RNA [Ringold, G. M., Yamamoto, K. R., Tomkins, G. M., Bishop, J. M. and Varmus, H. E. (1975) Cell 6, 299-305].