Pretreatment of HeLa S3 cells with 5 mM sodium n-butyrate markedly enhances cellular responsiveness to the synthetic glucocorticoid dexamethasone, using increased alkaline phosphatase activity as a marker for steroid action. In contrast, dexamethasone pretreatment does not affect the responses of cells to butyrate. Maximal effects of butyrate on steroid responsiveness occur after 2 days of pretreatment. The increased responsiveness of butyrate-pretreated cells to dexamethasone is partially explained by the collection of most cells at a block point in the hormonally responsive portion of the G1 phase of the cell cycle. Cell cycle population effects on steroid responsiveness are lost only gradually over 40 h after the release from butyrate, as cells leave the hormonally responsive late G1 and S phases. In addition to cell cycle population effects, a second, more rapidly reversible effect of butyrate on steroid responsiveness occurs within the late G1 phase itself at the butyrate block point. This second effect is fully and rapidly lost within 10 h after butyrate's removal, a time before the entry of the released cells into S phase. The reversal of butyrate-induced histone hyperacetylation was examined during this 10-h period. Hyperacetylation is lost in less than 2.5 h after butyrate's removal, suggesting that a rapidly reversible enhancement of glucocorticoid action may occur in the late G1 phase when histones are hyperacetylated. This rapidly reversible process appears to be distinct from the more slowly reversible cell cycle population effects.