Tumor cell resistance to many chemotherapeutic agents, including alkylating agents, cisplatin, and doxorubicin, is frequently associated with increased intracellular levels of the nonprotein sulfhydryl glutathione (GSH). Recent evidence has demonstrated that increased GSH levels can be accompanied by an increase in the activity of gamma-glutamylcysteine synthetase (GCS), which catalyzes the rate-limiting step in de novo synthesis of GSH, and by an increase in the steady state level of mRNA for the catalytic subunit of GCS. Using melphalan-resistant DU 145/M4.5 human prostate carcinoma cells, which express elevated GSH levels, GCS enzyme activity, and GCS mRNA levels, we sought to determine the mechanism(s) responsible for the increased GCS mRNA expression. As determined by Northern analyses and RNase protection assays, the steady state level of GCS message in the resistant cells was increased 10-20-fold, in comparison with the drug-sensitive parent DU 145 cells. No significant difference in gene copy number or evidence of rearrangement was detected in the resistant cell line by Southern analyses. The GCS-specific mRNA isolated from the resistant cells was less stable than that isolated from the drug-sensitive cells (half-lives of 6 hr and 9 hr, respectively), indicating that this difference does not contribute to the increased steady state levels in the resistant cells. Nuclear run-on experiments revealed that the GCS transcription rate in the DU 145/M4.5 cells was increased approximately 12-fold, in comparison with that detected in the DU 145 cells. This difference in transcription rate was comparable in magnitude to the difference in steady state mRNA levels detectable in the two cell populations. Similar correlations between steady state GCS mRNA levels and transcription rates were also observed in other DU 145 lines expressing intermediate degrees of resistance to melphalan and correspondingly intermediate GCS mRNA elevations. These data suggest that GCS expression is transcriptionally regulated in these melphalan-resistant tumor cells.