The environment-sensitive fluorescent probes provide excellent tools for studying membranes in their native state. We have modified the BODIPY-based fluorescent molecular rotor by increasing the number of alkyl moieties from one to two or three to achieve a more defined and deeper positioning of the probe in membranes. Detailed characterisation of fluorescence properties and localisation/orientation of probes was performed using a variety of fluorescence techniques and model membranes composed of different lipids. As expected, additional alkyls attached to the fluorophore moiety led to a deeper and more defined localisation of the probe in the lipid bilayer. The results strongly indicate that fluorescence properties of such probes are influenced not only by lipid packing but also by the orientation of the probe in membranes. The orientation of rotors studied herein was significantly altered by changes in the lipid composition of membranes. Our observations demonstrate the limits of BODIPY-based molecular rotors as environmental sensors in cellular membranes with complex lipid composition. The results presented herein also underline the importance of the detailed characterisation of fluorescent membrane dyes and provide a guide for future testing.