Seven cytologically distinct stages during micro spore development were identified and used to define the activation of an Arabidopsis anther-specific gene (apg) in transgenic tobacco plants containing an apg promoter-gus fusion. Histochemical analysis of GUS activity showed that the apg promoter was activated in miduninucleate microspores prior to equatorial and polar nuclear migration. Quantitative analysis in isolated spores showed that GUS activity per milligram of protein decreased progressively during pollen development, but on a per spore basis showed a second peak of activity in mid-bicellular pollen. Activation of the apg promoter in isolated gametophytic cells during development was also investigated following DNA delivery by microprojectile bombardment. Levels of transient expression were detectable in uninucleate microspores, but peaked in mid-bicellular pollen, in contrast to the progressive increase in activity of the promoter of the lsquolatersquo pollen gene lat52. These data show that the apg promoter is activated in a biphasic pattern, and indicate that the activity of transcription factors which mediate apg promoter activity persist through microspore mitosis, but decrease during pollen maturation.