Guard cells are specialized and metabolically active cells which arise during the differentiation of the epidermis. Using Nicotiana glauca epidermal peels as a source of purified guard cells, we have constructed a cDNA library from guard cell RNA. In order to isolate genes that are predominantly expressed in guard cells, we performed a differential screen of this library, comparing the hybridization of a radiolabeled cDNA probe synthesized from guard cell RNA to that from a mesophyll cell cDNA probe. Sixteen clones were isolated based on their greater level of hybridization with the guard cell probe. Of these, eight had high homology to lipid transfer protein (LTP), two were similar to glycine-rich protein (GRP), and one displayed high homology to proline-rich proteins from Arabidopsis thaliana (AtPRP2, AtPRP4) and from potato guard cells (GPP). Northern analysis confirmed that one or more NgLTP genes, NgGRP1, and NgGPP1 are all differentially expressed, with highest levels in guard cells, and low or undetectable levels in mesophyll cells and in roots. In addition, all are induced to some degree in drought-stressed guard cells. NgLTP and NgGRP1 expression was localized by in situ hybridization to the guard cells and pavement cells in the epidermis. NgGRP1 expression was also detected in cells of the vasculature. Genomic Southern analysis indicated that LTP is encoded by a family of highly similar genes in N. glauca. This work has identified members of a subset of epidermis- and guard cell-predominant genes, whose protein products are likely to contribute to the unique properties acquired by guard cells and pavement cells during differentiation.