The glutathione S-transferase pi gene (GST pi) is highly expressed in estrogen receptor negative (ER-) but not expressed in ER+ human breast cancer cell lines. To define regulatory mechanisms of GST pi gene expression, we analyzed both the activity of the GST pi promoter and the posttranscriptional fate of GST pi RNA sequences in three ER+ and three ER- breast cancer cell lines. Expression of a transiently transfected CAT reporter gene driven by the GST pi promoter and 2203 nucleotides of 5'-flanking sequences were similar in all six cell lines regardless of ER status. Endogenous GST pi transcription rates in nuclei isolated from ER- cells were quite low despite high steady state levels of cytoplasmic mRNA. Furthermore, the endogenous GST pi gene was transcribed in ER+ nuclei at rates similar to those obtained in ER- nuclei. We determined the stabilities of mRNAs transcribed from the endogenous GST pi gene (ER- cells) and from a stably transfected GST pi cDNA expression vector (ER+ and ER- cells). The endogenous GST pi mRNA was extraordinarily stable in ER- cells. Comparisons between transfected ER+ and ER- cells revealed no significant differences in the stabilities of transfection-derived GST pi mRNA sequences. We conclude that GST pi mRNA stability contributes significantly to the high levels of cytoplasmic mRNA observed in ER- cells, but that the differential expression of GST pi in ER+ versus ER- cells is governed by other posttranscriptional processes.