The interaction of biofuel sprays from an outward opening hollow cone injector and the flow field inside an internal combustion engine is analyzed by Mie-Scattering Imaging (MSI) and high-speed stereoscopic particle-image velocimetry (stereo-PIV). Two fuels (ethanol and methyl ethyl ketone (MEK)), four injection pressures (50, 100, 150, and 200 bar), three starting points of injection (60°, 277°, and 297° atdc), and two engine speeds (1,500 rpm and 2,000 rpm) define the parameter space of the experiments. The MSI measurements determine the vertical penetration length and the spray cone angle of the ethanol and MEK spray. Stereo-PIV is used to investigate the interaction of the flow field and the ethanol spray after the injection process for a start of injection at 60° atdc. These measurements are compared to stereo-PIV measurements without fuel injection performed in the same engine .The results of the measurements of the vertical penetration length show the flow field to strongly influence the spray behavior. Especially for late injection during the compression phase, a strong interaction of the tumble vortex and the spray is visible, such that liner and piston wetting effects are reduced. Compared to ethanol, MEK shows lower piston and liner wetting effects and a reduced influence of the flow field on the injection behavior. The measurements of the early spray cone angle evidence a decreasing influence of the flow field with increasing spray Reynolds number.The stereo-PIV measurements show a small change of the tumble trajectory through the fuel injection such that at late crank angles the tumble moves below the intake valves. Furthermore, a strong decrease in the spatially averaged out-of-plane vorticity shortly after injection occurred. The analysis of the kinetic and turbulent kinetic energy reveals lower values during the engine cycle with fuel injection. Moreover, at the end of the compression stroke the turbulent kinetic energy as well as the out-of-plane vorticity level is comparable with the measurements without injection.