The influence of 2,4-dichlorophenoxyacetic acid (2,4-D) on embryo-like structures (ELS) and plant development from barley microspores was determined. Microspores cultured on filters enabled simple modification of growth regulator concentrations. Regeneration frequencies obtained with 2,4- D as growth regulator were similar to the results achieved with the generally applied cytokinin 6-benzylaminopurine. If 2,4-D was applied after a regular mannitol pretreatment, maximal plant regeneration was achieved if 10-6 mol/L 2,4-D was present continuously or for 7 days. Alternatively, maximal plant formation was induced if 10-4 or 10-5 mol/L 2,4-D was present for 1 h or if present resp. 3 days or 1 day. Induction of plant regeneration by a l h treatment with 10-4 mol/L 2,4-D is a more generally observed phenomenon for single cells or small cell clusters of both dicotyledonous and monocotyledonous species. Without mannitol pretreatment, it was possible to induce plant production after 2,4-D treatment only in anther cultures. Without mannitol pretreatment no embryogenic type of microspores could be recognized at the moment of microspore isolation, and plating efficiency never reached 1%. In anther culture without mannitol pretreatment, a higher molarity and/or longer presence of 2,4-D was required and resulted only in about 1 green plant per anther. After application of a mannitol pretreatment, plant production increased at least 10 times. To our knowledge this is the first report on microspore-derived barley plants via androgenesis without any pretreatment. The combination of 2,4-D and anther pretreatment with mannitol as trigger for microspore differentiation is discussed.