We utilized a new cell ablation strategy to ablate specific anther cell types involved in the dehiscence process. The tobacco TA56 gene promoter is active within the circular cell cluster, stomium, and connective regions of the anther at different developmental stages. We introduced a cytotoxic TA56/barnase gene into tobacco plants together with three different anticytotoxic barstar genes. The anticytotoxic barstar genes were used to protect subsets of anther cell types from the cytotoxic effects of the TA56/barnase gene. The chimeric barstar genes were fused with (1) the tobacco TP12 gene promoter that is active at high levels in most anther cell types; (2) the soybean lectin gene promoter that is active earlier in the connective, and at lower levels in the circular cell cluster and stomium, than is the TA56 promoter; and (3) the tobacco TA20 gene promoter that is active at high levels in most anther cell types but has a different developmental profile than does the TP12 promoter. Normal anther development and dehiscence occurred in plants containing the TA56/barnase and TP12/barstar genes, indicating that barstar protects diverse anther cell types from the cytotoxic effects of barnase. Anthers containing the TA56/barnase and lectin/barstar genes also developed normally but failed to dehisce because of extensive ablation of the circular cell cluster, stomium, and contiguous connective regions. Anthers containing the TA56/barnase and TA20/barstar genes failed to dehisce as well. However, only the stomium region was ablated in these anthers. The connective, circular cell cluster, and adjacent wall regions were protected from ablation by the formation of barnase/barstar complexes. We conclude that anther dehiscence at flower opening depends on the presence of a functional stomium region and that chimeric barnase and barstar genes containing promoters that are active in several overlapping cell types can be used for targeted cell ablation experiments.