We have used reverse-genetic analysis to investigate the function of MAP3K epsilon 1 and MAP3K epsilon 2, a pair of closely related Arabidopsis thaliana genes that encode protein kinases. Plants homozygous for either map3k epsilon 1 or map3k epsilon 2 displayed no apparent mutant phenotype, whereas the double-mutant combination caused pollen lethality. Transmission of the double-mutant combination through the female gametophyte was normal. Tetrad analysis performed using the Arabidopsis quartet mutation demonstrated that the pollen-lethal phenotype segregated at meiosis with the map3k epsilon 1;map3k epsilon 2 genotype. We used transmission electron microscopy to determine that double-mutant pollen grains develop plasma membrane irregularities following pollen mitosis I. Analysis of the subcellular localization of a yellow fluorescent protein (YFP):MAP3Kepsilon1 fusion protein using confocal microscopy and biochemical fractionation indicated that a substantial portion of the MAP3Kepsilon1 present in Arabidopsis cells is localized to the plasma membrane. Taken together, our results suggest that MAP3Kepsilon1 is required for the normal functioning of the plasma membrane in developing Arabidopsis pollen.