The stress-induced hyperthermia (SIH) paradigm in group-housed mice allows screening of putative anxiolytic drugs. The group-housed SIH was adapted to singly housed animals in order to drastically reduce the number of animals used. The effect of various stressors on rectal temperature was measured in order to find a simple and reliable test procedure. Repeated, but not single disturbance of animals resulted in a strong hyperthermia (deltaT) within 10 min. Similar hyperthermic responses were obtained after immobilization for 1 min or rectal temperature measurement itself. Neither a 120 dB acoustic stimulus, nor repeated 1 mA footshocks led to a temperature change, but 2 mA electric footshocks led to hyperthermia. The final test paradigm chosen involved repeated temperature measurement at a 10 min interval, thus providing both information on basal temperature and deltaT in each animal within a short time frame. Repeated temperature measurements at 10 min intervals revealed a maximum hyperthermia after approximately 30 min, but up to 70% of the hyperthermia is already present 10 min after the first measurement. Repeated use of animals at successive daily or weekly intervals resulted in a gradual increase of both the basal temperature and the temperature 10 min later. At short inter-test intervals (one day) deltaT also decreased, whereas weekly intervals did not affect the amplitude of deltaT. Prior injection of the animals resulted in modest hyperthermia, that returned to baseline after 60 min. The anxiolytics diazepam and 5-HT1A receptor agonist flesinoxan dose-dependently suppressed SIH. The antidepressant amitriptyline lowered temperature levels but did not affect deltaT. The SIH model in singly housed mice appears a fast and reproducible screening test for anxiolytic activity. Compared to the group-housed version, the singly-housed SIH enabled a drastic reduction in the number of animals used.