The Arabidopsis thaliana MYB26/MALE STERILE35 (MS35) gene is critical for the development of secondary thickening in the anther endothecium and subsequent dehiscence. MYB26 is localized to the nucleus and regulates endothecial development and secondary thickening in a cell-specific manner in the anther. MYB26 expression is seen in anthers and also in the style and nectaries, although there is no effect on female fertility in the ms35 mutant. MYB26 expression in anthers occurs early during endothecial development, with maximal expression during pollen mitosis I and bicellular stages, indicating a regulatory role in specifying early endothecial cell development. Overexpression of MYB26 results in ectopic secondary thickening in both Arabidopsis and tobacco (Nicotiana tabacum) plants, predominantly within the epidermal tissues. MYB26 regulates a number of genes linked to secondary thickening, including IRREGULAR XYLEM1 (IRX1), IRX3, IRX8, and IRX12. Changes in expression were also detected in two NAC domain genes, NAC SECONDARY WALL-PROMOTING FACTOR1 (NST1) and NST2, which have been linked to secondary thickening in the anther endothecium. These data indicate that MYB26 regulates NST1 and NST2 expression and in turn controls the process of secondary thickening. Therefore, MYB26 appears to function in a regulatory role involved in determining endothecial cell development within the anther and acts upstream of the lignin biosynthesis pathway.