We report here a new selectable marker for tobacco immature pollen transformation based on the expression of dihydrofolate reductase (dhfr) gene which confers resistance to methotrexate (Mtx). Two immature pollen transformation approaches, i.e., male germ line transformation and particle bombardment of embryogenic mid-bicellular pollen have been used for the production of stable transgenic tobacco plants. In the first method, two methotrexate-resistant plants were selected from a total of 7161 seeds recovered after transformation experiments. In the second method, four methotrexate-resistant plants were obtained from 29 bombardments using 3.7 x 10(5) pollen grains per bombardment. Southern analysis confirmed the transgenic nature of T0 and T1 candidate transgenic plants, and a genetic analysis showed that the transgenes are transmitted to subsequent generations.