Sequencing of Arabidopsis thaliana genome paved the way for functional genomics studies. Aiming the functional characterization of genes with a major role in A. thaliana male reproductive development, we used DNA microarray analysis and T-DNA insertional mutants. A DNA chip containing all A. thaliana annotated genes was screened with probes corresponding to mRNAs of apetala3-3 and wild type early flower buds, allowing the identification of 139 genes repressed in ap3-3 flowers. Seventeen T-DNA tagged lines for genes encoding proteins related to signal transduction or transcription were obtained from Salk Institute Genomic Analysis Laboratory. T-DNA homozigous plants were obtained through segregation analysis and confirmed by PCR genotyping. A severely impaired male fertility phenotype was observed in Salk_022698 line, in which the T-DNA disrupts At2g40850, a gene encoding a putative phosphatidylinositol 3- and 4- kinase. Phosphatidylinositol kinases are involved in metabolism of phosphatidylinositol, a phospholipid crucial in several signaling pathways and reported to be involved in vesicle trafficking in both animals and higher plants. Phylogenetic analysis of rice and Arabidopsis PI3_PI4K proteins revealed a small family of 17 plant proteins, none of which functionally characterized yet. RT-PCR gene expression analysis showed that At2g40850 is highly expressed in inflorescences, root and leaves. Microscopic analysis of At2g40850 mutant stamen anatomy revealed tapetal cells with abnormal vacuoles at stage 7 of stamen development, what may be the cause of the defective pollen grains observed in anthers locules at stage 13. These results indicate that the partial male-sterility may be due to tapetum malfunction caused by lack of functional At2g40850 protein. At2g40850 cDNA was cloned and inserted, after DNA sequencing, in a GATEWAY cloning vector that was used to obtain constructs for three different binary vectors: overexpression (35S:At2g40850cDNA), translational fusion with GFP for protein subcellular localization (35S:At2g40850-GFP), and RNAi vector for gene silencing (RNAiAt2g40850). Wild type plants are being transformed with these three distinct constructs via Agrobacterium tumefaciens. We expect that, taken together, our results lead to the elucidation of At2g40850 protein function during microsporogenesis of A. thaliana.