Although computer-assisted sperm analysis (CASA) outperforms manual techniques, many investigators rely on non-automated analysis due to the high cost of commercial options. In this study, we have written and validated a free CASA software primarily for analysis of fish sperm. This software is a plugin for the free National Institutes of Health software ImageJ and is available with documentation at . That it is open source makes possible external validation, should improve quality control and enhance the comparative value of data obtained among laboratories. In addition, we have improved upon the traditional velocity straight line (VSL) algorithm, eliminating inaccurate characterization of highly curved fish sperm paths. Using this system, the motion of zebrafish (Danio rerio) sperm was characterized relative to time post-activation and the impact of acquisition conditions upon data analysis determined. There were decreases in velocity and path straightness (STR), but not linearity (LIN), relative to time. From 30 to 300 frames/s, frame rate significantly affected curvilinear velocity (VCL) and STR measurements. Sperm density in the field of view did not affect any measured parameter. There was significant inter-male variation for VCL, VSL, velocity average path (VAP), percent motility, path character (STR, LIN), and duration of motility. Furthermore, relative sperm output (a measure reflecting both semen volume and concentration) was positively correlated to percent motility. For all motion parameters measured (except duration), the average CV was < or =10%, comparable to values obtained using commercial systems.