Sperm motility, crucial for fertilization, has been mostly studied in two dimensions (2D) by recording their swimming trajectories near a flat Surface. However, spermatozoa swim in three-dimensions (3D) to find eggs, with their speed being the main impediment to track them under realistic conditions. Here, we describe a novel method allowing 3D tracking and analysis of the trajectories of multiple free-swimming sperm. The system uses a piezo-electric device displacing a large focal distance objective mounted on a microscope to acquire 70 image stacks per second, each stack composed of 60 images that span a depth of 100 pro. With this method, 3D paths of multiple sperm in the same field could be visualized simultaneously during 1 s. Within the same sample we found that surface-confined sperm swam 25% slower, produced 3-fold fewer circular revolutions per second, and had trajectories of 134% greater radius of curvature than those sperm swimming freely in 3D. (C) 2008 Elsevier Inc. All rights reserved.