We have analysed the promoter regions of two closely related auxin-regulated glutathione S-transferase genes. All active deletion constructs tested showed expression of the reporter gene beta-glucuronidase (gusA) in root tips of young seedlings and newly developing lateral roots. Auxin treatment greatly enhanced the level of expression. The Nt103-1 promoter region -370/-276 was found to be necessary, at least as a quantitative element to confer auxin-responsiveness to a reporter gene, and sequences responsible for the auxin-responsiveness must be located downstream of -370. The region -651/-370 contains sequence information necessary for uninduced expression. The Nt103-35 promoter manifested its auxin-responsiveness within the -504/-310 region. Electrophoretic mobility shift analysis, using nuclear extracts from tobacco leaves and suspension cells, identified a factor binding to a sequence (ap103, TGAGTCT) at position -560 of the Nt103-1 promoter, which shows homology to the mammalian AP-1 site. A second factor was found to bind a sequence (as103, ATAGCTAAGTGCTTACG) with homology to the CaMV 35S promoter as-1 element. The as103 element is present in both promoters and positioned around -360, so within the region determined to be indispensable for the response to auxin. A third factor was found binding to the -276/-190 region of both promoters. Combined, these data point to the relevance of a 90 bp region for auxin-induced activity of both tobacco genes. The ASF-1 like factor binding to the as103 element within this region might be involved in mediating the auxin response.