The automated CellSoft semen analyzer identifies human spermatozoa on the basis of user-defined values for cell size and luminosity. Previous studies have shown that the non-sperm particles usually present in seminal plasma would interfere with the computerized determination of sperm concentration by the CellSoft system. Therefore, an effective method to separate the sperm from non-sperm particles would be desirable to obtain accurate determination of sperm concentration. In the present study, sperm concentrations in 72 semen samples before and after discontinuous Percoll gradient centrifugation were determined by the automated CellSoft system and the results compared with those obtained with the routine procedure using the haemocytomer according to the World Health Organization laboratory manual. The computerized measurement caused a significant overestimation when the sperm concentration in semen was less than 80 x 10(6)/ml. Processing of human semen sample by the simple two-layer discontinuous Percoll gradient centrifugation removed the majority of non-sperm particles and the overestimation by the automated CellSoft system became significant only when the sperm concentration in the final Percoll sperm preparation was less than 10 x 10(6)/ml. These findings indicate that the automated CellSoft semen analyzer has to be improved to allow for the correction of non-sperm particles in seminal plasma or processed sperm samples before it can be used to provide sufficiently accurate sperm concentration results for routine laboratory service or research purposes.