Genetic engineering is becoming a useful tool in the improvement of plants and plant-based raw materials. Varieties with value-added traits are developed for nonfood use in industrial and medical production, and different production lines must be kept separate. For good management practices, knowledge of relevant gene flow parameters is required. In the present study, pollen-mediated dispersal of transgenes via cross-fertilization was examined. A transgenic barley (Hordeum vulgare L.) line carrying a marker gene coding for neomycin phosphotransferase II (nptII) was used as a pollen donor. For maximum resolution, a cytoplasmically male-sterile barley line was utilized as recipient and the flow of nptII transgene was monitored at distances of 1, 2, 3, 6, 12, 25, 50, and 100 m from the donor plots of 225 and 2000 m2. Male-fertile plots at a distance of 1 m were included to measure the transgene flow in normal barley. The number of seeds obtained from male-sterile heads diminished rapidly with distance and only a few seeds were found at distances of 50 and 100 m. Molecular genetic analysis (polymerase chain reaction-PCR) revealed that all seeds obtained from male-sterile heads at a distance of 1 m were transgenic, as anticipated. However, only 3% of the distant seeds (50 m) actually carried the transgene, whereas most of them resulted from fertilization with nontransgenic background pollen. This background pollen was mainly due to pollen leakage in some male-sterile heads. In normal male-fertile barley, the cross-fertilization frequency with transgenic pollen varied from 0 to 7% at a distance of 1 m, depending on weather conditions on the heading day. We conclude that, because of competing self-produced and nontransgenic background pollen, the possibility of cross-pollination is very low between a transgenic barley field and an adjacent field cultivated with normal barley. However, adequate isolation distances and best management practices are needed for cultivation of transgenic barley.