We provide a basis for automated single-cell sorting based on optical trapping and manipulation using human peripheral blood as a model system. A counterpropagating dual-beam optical-trapping configuration is shown theoretically and experimentally to be preferred due to a greater ability to manipulate cells in three dimensions. Theoretical analysis performed by simulating the propagation of rays through the region containing an erythrocyte (red blood cell) divided into numerous elements confirms experimental results showing that a trapped erythrocyte orients with its longest axis in the direction of propagation of the beam. The single-cell sorting system includes an image-processing system using thresholding, background subtraction, and edge-enhancement algorithms, which allows for the identification of single cells. Erythrocytes have been identified and manipulated into designated volumes using the automated dual-beam trap. Potential applications of automated single-cell sorting, including the incorporation of molecular biology techniques, are discussed. © 2001 Society of Photo-Optical Instrumentation Engineers.