The World Health Organization suggests that 'one-step' type disposable chambers lack the accuracy and precision of the haemocytometer method for assessing sperm concentration. The purpose of this particular study was to compare sperm concentration measurements and motility using the Neubauer((R)) haemocytometer with those obtained using three 'one-step' methods: the Microcell((R)) slide, Leja((R)) slide and a plain glass slide with a 22x22 mm coverslip. A total of 200 sperm concentration measurements and 100 motility assessments were performed on all chambers. Paired comparisons showed enormous discrepancies between the counts, particularly between the Neubauer((R)) and other chambers (P < 0.0001). This discrepancy was less pronounced in oligozoospermic samples, and samples with low (<30% progression) motility but more pronounced in normozoospermic samples and those with good motility (>50% progression). In addition, concentration assessments from a fresh undiluted and unfixed semen sample on the Microcell((R)) slide were found to be significantly lower than both fixed counts on the same slide (P = 0.011) and the initial laboratory reading on the Neubauer((R)) chamber (P = 0.009). No differences were observed in progressive motility between the different chambers and a plain glass slide. There appears to be little comparison between the haemocytometer and either re-useable or disposable one-step chambers. The unfortunate consequence of this is that diagnostic semen analysis and guidelines for allocation of patients to appropriate treatment groups will vary from centre to centre, depending on the method used and may, on occasion, be erroneous.