Staining of cells with acridine orange (AO) has been widely accepted as a predictor of DNA damage in many cell types. Because of variability of protocols used in previous studies, the AO staining technique has not been widely accepted as a screening test to predict DNA damage in human sperm. In order to further validate the use of AO staining, sperm were evaluated using numerous variations in the staining protocol. This study also elucidated the effects of cryopreservation on sperm DNA. Sperm fixation in Carnoy's solution showed significantly (P < 0.05) more DNA damage (29.9 +/- 4.5%) than 2% glutaraldehyde (14.4 +/- 2.1%), 4% paraformaldehyde (5.5 +/- 1.7%), no fixation (15.8 +/- 4.3%) but did not differ from Diff Quik solution (19.2 +/- 5.8%). No difference was observed for sperm DNA damage assessment using a 0.2 m (15.5 +/- 3.2%) or 0.3 m (14.9 +/- 3.3%) concentration of Na(2)HPO(4).7H(2)O in the AO staining solution. Frozen-thawed semen samples showed increased damage to sperm DNA under both Carnoy's (fresh: 10.9 +/- 1.3%; frozen: 30.8 +/- 2.9%; P < 0.05) and Diff Quik fixation (fresh: 6.2 +/- 0.8; frozen: 17.1 +/- 2.5%P < 0.05). Present data also showed that spermatozoa from some individuals are more prone to DNA damage after freezing and thawing procedures than others. In conclusion, Carnoy's fixative provides a better predictive value for DNA damage to sperm using AO staining. Additionally, cryopreservation increased damage to the sperm DNA.