SummaryThe effect of a commonly used anaesthetic, ketamine/xylazine and/or carbon dioxide (CO 2 ) on plasma luteinizing hormone releasing hormone (LHRH) and testosterone concentrations was determined in male Sprague-Dawley rats. These values were compared with values obtained from pre-anaesthetic control samples. Ketamine/xylazine treatment did not significantly affect testosterone concentrations. In contrast, LHRH started to decrease one hour after ketamine/xylazine administration and continued to significantly decrease after 24 h. In addition, in the CO 2 euthanasia-only group, LHRH concentrations were also significantly decreased. These results suggest that ketamine/xylazine anaesthesia followed by CO 2 euthanasia 24 h later is exerting a significant effect on LHRH concentrations 24 h after anaesthetizing, while only having a slight effect on testosterone, and that CO 2 is exerting an immediate significant effect on LHRH. In conclusion, LHRH analysis should be avoided after ketamine/xylazine anaesthesia and CO 2 euthanasia.