The interactions between single molecules and three-dimensional donut modes in fluorescence microscopy are discussed based on the vector diffraction theory of light. We find that the use of donut modes generated from a linearly polarized laser beam can yield information about the orientation of immobilized single molecules, allowing for their use in orientational imaging. While fairly insensitive over a range of orientations, this technique is seen to be very sensitive for the subset of orientations where the transition dipole of the molecule is oriented close to the optical axis of the microscope and perpendicular to the input polarization. In a second part of the paper we discuss the impact of the molecular orientation on the resolution improvement in STED microscopy. We find that, even for circularly polarized excitation light, the expected resolution improvement depends on the orientation of the molecule relative to the optical axis of the microscope. (c) 2007 Optical Society of America.