We have characterized cotton (Gossypium hirsutum L.) genes encoding type 1 metallothionein-like proteins that are highly expressed in roots. Little or no expression of these genes was detected in other organs and tissues. The deduced amino acid sequences have a high degree of similarity with type 1 metallothionein-like proteins from other plants, including a central hydrophobic domain flanked by conserved cysteine-rich motifs. The type 1 metallothionein-like genes of cotton are encoded by a small gene family. One gene (MT1-A) was analyzed in detail and found to have three exons which are 52, 83 and 397 bp long, and two introns 130 and 1042 bp in length. Three of the type 1 metallothionein-like genes are organized in a tandom array, and the 5'-flanking regions of these genes share a high degree of sequence similarity. Two of the clustered genes (MT1-A and MT1-B) are expressed at about equal levels in roots and use the same transcription start site. A 640 bp promoter fragment from the MT1-A gene was sufficient to direct expression of beta-glucuronidase (GUS) in transformed cotton roots. The expression was highest near the root tip.