Presence of proteins in the surface wax of plants has been noted but no protein has been identified heretofore. A 9-kDa protein was purified as the major protein from the surface wax of broccoli (Brassica oleracea var. italica) leaves and the N-terminal amino acid sequence was determined. A cDNA corresponding to the wax-associated protein, designated WAX9, was sequenced revealing an open reading frame that codes for an 11.9-kDa polypeptide of 118 amino acids including a 25-amino acid leader peptide. The amino acid sequence showed 40 to 50% identity with nonspecific lipid transfer proteins isolated from various plants. Northern blots showed that a wax9 transcript of 650 nucleotides was highly expressed in leaves, stems, and flower buds and only at a reduced level in open flowers and not in roots. The transcript level decreased as the leaf fully expanded. The results suggest that the wax9 gene is expressed at the highest level at the time of maximal synthesis of the surface wax. Immunogold labeling studies showed that the WAX9 protein was present mainly in the cell wall of the epidermis and mesophyll tissues as well as in the phloem. However, immunological measurements showed that 50 and 4% of the total WAX9 was in the surface wax in the young and old leaves, respectively. Since immunogold labeling method would involve the loss of the surface wax, it would not detect the WAX9 protein in the wax. Thus, the present results reveal for the first time that a major portion of lipid transfer protein is in the surface wax.