We study the fluorescence patterns produced by single CdSe nanocrystals in defocused microscopy. The patterns are directly related to the structure of the nanocrystal emission dipole and their analysis provides a full determination of the three-dimensional orientation of the nanoparticles. In the case of a high numerical aperture objective, we show that the contour intensities of the defocused patterns can be fitted by a simple analytical formula. Finally, we demonstrate that defocused imaging can be used to track both the position and the orientation of single CdSe nanocrystals coupled to membrane receptors in live cells. (c) 2005 Elsevier B.V. All rights reserved.