The frequency of lateral root initiation in tomato (Lycopersicon esculentum Mill cv. VFN8) seedling roots is increased over eightfold in response to 1.6 microM alpha-naphthalene-acetic acid (NAA). To identify genes that are activated during lateral root initiation, a cDNA library was made with RNA from roots treated with auxin and differentially screened with radioactive probes made from RNA isolated from treated and untreated roots. A cDNA clone, TR132, was identified that hybridized to a transcript that was induced within 4 h of auxin treatment and increased tenfold by 72 h. A gene (RSI-1) corresponding to the TR132 cDNA was cloned and characterized with regard to its nucleotide sequence, transcription start site and chromosomal map position. Approximately 1 kb of the 5' flanking DNA was linked to the beta-glucuronidase (GUS) protein coding region and tested for expression in transgenic tomato seedlings. GUS activity was observed in both lateral and adventitious root initials, including very early initials, and persisted until shortly after the lateral emerged from the parent tissue. In roots from seedlings with high activity, GUS expression was also observed in the root cap and vascular tissue. The predicted RSI-1 protein is rich in cysteine, lysine and proline, and includes an N-terminal region with characteristics of a signal peptide. The putative mature protein exhibits 79% amino acid identity to a protein encoded by a gene (GAST1) that is induced by gibberellic acid in tomato shoots.