As a strategy for the identification of T-DNA-tagged gametophytic mutants, we have used T-DNA insertional mutagenesis based on screening for distorted segregation ratios by antibiotic selection. Screening of approximately 1000 transgenic Arabidopsis families led to the isolation of eight lines showing reproducible segregation ratios of approximately 1:1, suggesting that these lines are putative gametophytic mutants caused by T-DNA insertion at a single locus. Genetic analysis of T-DNA transmission through reciprocal backcrosses with wild type showed severe reductions in genetic transmission of the T-DNA through the male and/or female gametes. Direct evidence for mutant phenotypes in these lines was investigated by DAPI staining of mature pollen grains and by the analysis of seed set and embryo sac morphology in cleared ovules. One line, termed limpet pollen, showed a novel pollen phenotype in that the generative cell failed to migrate inward after pollen mitosis I, such that the generative or sperm cells remained against the pollen wall. Two other lines, andarta and tistrya, were defective in female transmission and showed an early arrest of embryo sac development with the viable megaspore not initiating the nuclear division cycles. These data demonstrate the efficacy of a segregation ratio distortion strategy for the identification of T-DNA-tagged gametophytic mutants in Arabidopsis.