Quantitative microscopy has been extensively used in biomedical research and has provided significant insights into structure and dynamics at the cell and tissue level. The entire procedure of quantitative microscopy is comprised of specimen preparation, light absorption/reflection/emission from the specimen, microscope optical processing, optical/electrical conversion by a camera or detector, and computational processing of digitized images. Although many of the latest digital signal processing techniques have been successfully applied to compress, restore, and register digital microscope images, automated approaches for recognition and understanding of complex subcellular patterns in light microscope images have been far less widely used. We describe a systematic approach for interpreting protein subcellular distributions using various sets of subcellular location features (SLF), in combination with supervised classification and unsupervised clustering methods. These methods can handle complex patterns in digital microscope images, and the features can be applied for other purposes such as objectively choosing a representative image from a collection and performing statistical comparisons of image sets. © 2004 Society of Photo-Optical Instrumentation Engineers.