Animal and care use practices are constantly evolving. These can have unexpected consequences on the data collected from such procedures. One example is the recent change in our animal facility, based on recommendations from the Newcastle Consensus Meeting on Carbon Dioxide Euthanasia of Laboratory Animals, from CO(2) to isoflurane for anesthesia. The current study was conducted to determine the effects of isoflurane on sperm motility, as compared to two different CO(2) euthanasia procedures. Sperm motility was evaluated after euthanasia by a standard 5-minute CO(2) euthanasia procedure, an extended 10-minute CO(2) euthanasia procedure, or by isoflurane anesthesia followed by exsanguination (iso/exsanguination). The 5-minute CO(2) procedure produced sperm motility of 94.3 ± 1.7% motile sperm with 65.6 ± 16.8 sperm/field. By comparison, iso/exsanguination reduced that count to 3.3 ± 2.3 sperm/field and only 60.7 ± 32.0% motile sperm. The reduction in sperm motility after iso/exsanguination appeared to have been due primarily to the reduction in the number of sperm expelled from the vas deferens (3.3), compared to that after 5-minute CO(2) (65.6). This reduction in number of sperm available for evaluation, in the presence of a constant level of background debris, which was counted by the computer optics system as nonmotile sperm, resulted in an apparent reduction in motility. Using the extended 10-minute CO(2) procedure produced sperm data in between the other two extremes: 77.6 ± 36.1% motile sperm with 34.6 ± 28.3 sperm/field. The results of this study support the hypothesis that isoflurane inhibits contraction of the smooth muscle of the vas deferens, resulting in a decreased number of expelled sperm. Given these findings, it is important that careful consideration be taken to select an appropriate anesthesia/euthanasia method.