Glutathione S-transferases (GSTs) play major roles in the protection of plants from biotic and abiotic stresses through the detoxification of xenobiotics and toxic endogenous products. This report describes additional complexity in the regulation of the well characterized stress-responsive Arabidopsis thaliana GSTF8 promoter. This complexity results from the use of multiple transcription start sites (TSS) to give rise to alternate GSTF8 transcripts with the potential to produce two in-frame proteins differing only in their N-terminal sequence. In addition to the originally mapped TSS (Chen, W., Chao, G., and Singh, K. B. (1996) Plant J. 10, 955-966), a further nine TSS have been identified, with the majority clustered into a distinct group. The most 3' TSS gives rise to the major message (GSTF8-S) and the shorter form of the protein, whereas those originating from upstream TSS (GSTF8-L) are more weakly expressed and encode for the larger form of the protein. Differential tissue-specific and stress-responsive expression patterns were observed (e.g. GSTF8-L is more highly expressed in leaves compared with roots, whereas GSTF8-S expression has the opposite pattern and is much more stress-responsive). Analysis of GSTF8-L and GSTF8-S proteins demonstrated that GSTF8-L is solely targeted to plastids, whereas GSTF8-S is cytoplasmic. In silico analysis revealed potential conservation of GSTF8-S across a wide range of plants; in contrast, conservation of GSTF8-L was confined to the Brassicaceae. These studies demonstrate that alternate TSS of the GSTF8 promoter are used to confer differential tissue-specific and stress-responsive expression patterns as well as to target the same protein to two different subcellular localizations.