Upstream sequences of the H4 histone gene FO108 located between nt –418 to –213 are stimulatory for in vivo transcription. This domain contains one protein/DNA interaction site (H4‐Site III) that binds factor H4UA‐1. Based on methylation interference, copper‐phenanthroline protection, and competition assays, we show that H4UA‐1 interacts with sequences between nt −345 to −332 containing an element displaying sequence‐similarity with the thyroid hormone response element (TRE). Using gel retardation assays, we also demonstrate that H4UA‐1 binding activity is abolished at low concentrations of Zn2+ (0.75 mM), a characteristic shared with the thyroid hormone (TH) receptor DNA binding protein. Interestingly, phosphatase‐treatment of nuclear proteins inhibits formation of the H4UA‐1 protein/DNA complex, although a complex with higher mobility (H4UA‐1b) can be detected; both complexes share identical protein‐DNA contacts and competition behaviors. These findings suggest that phosphorylation may be involved in the regulation of H4‐Site III protein/DNA interactions by directly altering protein/protein associations. H4‐Site III interactions were examined in several cell culture systems during cell growth and differentiation. We find that H4UA‐1 binding activity is present during the cell cycle of both normal diploid and transformed cells. However, during differentiation of normal diploid rat calvarial osteoblasts, we observe a selective loss of the H4UA‐1/H4‐Site III interaction, concomitant with an increase of the H4UA‐1b/H4‐Site III complex, indicating modifications in the heteromeric nature of protein/DNA interactions during downregulation of transcription at the cessation of proliferation. Transformed cells have elevated levels of H4UA‐1, whereas H4UA‐1b is predominantly present in normal diploid cells; this alteration in the ratio of H4UA‐1 and H4UA‐1b binding activities may reflect deregulation of H4‐Site III interactions in transformed cells. We propose that H4‐Site III interactions may contribute, together with protein/DNA interactions at proximal regulatory sequences, in determining the level of H4‐FO108 histone gene transcription.