In cells, steroid hormone receptors interact with target enhancer elements on nucleosomes to regulate transcription of genes. To elucidate how nucleosomes can potentially regulate the interactions of steroid receptors with steroid response elements, we have examined the effects of nucleosome positioning and histone source on the binding of the progesterone receptor to DNA elements on nucleosomes reconstituted in vitro. We find that the affinity of the receptor for its response element is dependent on the position of the element within the nucleosome, but not on the histone source, active or inactive chromatin. Our results suggest that the strength of DNA-histone interactions within the nucleosome modulates the binding of progesterone receptor to response elements. Thus, nucleosome positioning is likely to influence the function of steroid receptors in vivo.