Although biofilms represent a common bacterial life style in clinically and environmentally important habitats, there is scant information on the extent of gene transfer in these spatially structured populations. The objective of this study was to gain insight into factors that affect transfer of the promiscuous multi-drug resistance plasmid pB10 in E. coli biofilms. Biofilms were grown in different experimental settings and plasmid transfer was monitored using laser scanning confocal microscopy and plate counting. In closed flow cells, plasmid transfer in surface-attached submerged biofilms was negligible. In contrast, high plasmid transfer efficiency was observed in a biofilm floating at the air-liquid interface in an open flow cell under low flow rates. A vertical flow cell and a batch culture biofilm reactor were then used to detect plasmid transfer at different depths away from the air-liquid interface. Extensive plasmid transfer only occurred in a narrow zone near that interface. The much lower transfer frequency in the lower zones coincided with rapidly decreasing oxygen concentrations. However, when an E. coli csrA mutant was used as recipient, a thick biofilm was obtained at all depths, and plasmid transfer occurred at similar frequencies throughout. These results and data from separate aerobic and anaerobic matings suggest that oxygen can affect IncP-1 plasmid transfer efficiency, not only directly but also indirectly through influencing population densities and therefore co-localization of donors and recipients. In conclusion, the air-liquid interface can be a hot-spot for plasmid-mediated gene transfer due to high densities of juxtaposed donor and recipients cells.