Fiber-specific genes are expressed preferentially or exclusively in cotton (Gossypium spp.) fiber and are thought to have important functions in fiber development. The promoters of these genes are of interest because they control transcription in the fiber cell and may be used in the genetic manipulation of fiber quality. The promoter of a cotton lipid transfer protein gene, FSltp4, was isolated and shown to direct fiber-specific transcription of an abundant mRNA in cotton. In transgenic tobacco, this promoter was strongly active in leaf trichomes. Deletion analysis of the promoter identified an AT-rich 84 bp fiber specificity region (FSR) necessary for activity exclusively in the fiber cells. Cotton fiber proteins that bind the FSR were isolated using a yeast one-hybrid assay. One of these was a putative AT-hook transcription factor (GhAT1) containing two AT-hook motifs. GhAT1 was shown to be nuclear localized, and GhAT1 transcripts were found to be preferentially expressed in ovules and non-fiber tissues. Overexpression of GhAT1 strongly repressed the activity of the FSltp4 promoter in the trichomes of transgenic tobacco. These results suggest that GhAT1 assists in the specification of fiber cells by repressing FSltp4 in the non-fiber tissues of the cotton plant.