The Arabidopsis thaliana PROTODERMAL FACTOR1 (PDF1) gene encoding a putative extracellular proline-rich protein is exclusively expressed in the L1 layer of shoot apices and the protoderm of organ primordia. In order to identify essential cis-regulatory sequences required for the L1 layer-specific expression, a series of 5' deletions of the PDF1 promoter were fused to the beta-glucronidase (GUS) gene and introduced into Arabidopsis plants. Our analysis revealed that the minimum region necessary to confer L1-specific expression of PDF1 is confined within a 260-bp fragment upstream of the transcription start site. We identified an 8-bp motif in this region that is conserved between promoter regions of all the L1-specific genes so far cloned, and we designated it the L1 box. Electrophoretic mobility shift assays demonstrated that the L1-specific homeodomain protein ATML1 can bind to the L1 box sequence in vitro. The GUS expression in transgenic plants disappeared when a mutation that abolishes binding of ATML1 was introduced into the PDF1 l1 box sequence of the construct. These results suggest that the L1 box plays a crucial role in the regulation of PDF1 expression in L1 cells and that ATML1 could cooperate to drive L1-specific expression.