We investigated the mechanism of sodium butyrate (NaB)-mediated induction of mdr1 mRNA in parental (wild type) and multidrug-resistant (Ad1000) SW620 colon cancer cell lines. NaB treatment resulted in reversible, time-dependent increases in nuclear run-on transcription of endogenous mdr1 in these cell lines that paralleled the reversible increases of mdr1 mRNA in both timing and magnitude. In contrast, NaB treatment had no effect on mdr1 mRNA stability. Thus, the effects of NaB on mdr1 mRNA levels are fully attributable to altered mdr1 transcription. Furthermore, NaB induces the expression of transiently transfected chloramphenicol acetyltransferase reporter plasmids that are under the transcriptional control of the mdr1 promoter (mdrCAT vectors). Transfections using mdrCAT vectors modified by deletion and site-directed mutagenesis of the mdr1 promoter indicate that NaB-mediated induction of these vectors is at least partially dependent upon sequences present in the basal mdr1 promoter between -89 and +11 relative to the start site of transcription. The Y-box motif located between -82 and -73 contributes to NaB inducibility of mdrCAT vector expression in Ad1000 SW620 cells.