We employed a gene trap approach to identify genes expressed in stomatal guard cells of Arabidopsis thaliana. We examined patterns of reporter gene expression in approximately 20,000 gene trap lines, and recovered five lines with exclusive or preferential expression in stomata. The screen yielded two insertions in annotated genes, encoding the CYTOCHROME P450 86A2 (CYP86A2) mono-oxygenase, and the PLEIOTROPIC DRUG RESISTANCE 3 (AtPDR3) transporter. Expression of the trapped genes in guard cells was confirmed by RT-PCR experiments in purified stomata. Examination of homozygous mutant lines revealed that abscisic acid (ABA)-induced stomatal closure was impaired in the atpdr3 mutant. In three lines, insertions occurred outside transcribed units. Expression analysis of the genes surrounding the trapping inserts identified two genes selectively expressed in guard cells, corresponding to a PP2C PROTEIN PHOSPHATASE and an unknown expressed protein gene. Statistical analyses of the chromosomal regions tagged by the gene trap insertions revealed an over-represented [A/T]AAAG motif, previously described as an essential cis-active element for gene expression in stomata. The lines described in this work identify novel genes involved in the modulation of stomatal activity, provide useful markers for the study of developmental pathways in guard cells, and are a valuable source of guard cell-specific promoters.