For the accurate functional analysis of the gas exchange in the lungs or evaluation of artificial oxygen carriers in spontaneously breathing anaesthetized small animals, we developed a new respiratory micro-valve. The body of the valve is made of aluminium, and the flaps are made of silicone rubber. The maximum flow rate in a rat measured with a pneumotachograph and the micro-valve was an average of 19.9 ml/s during inspiration, and 17.8 ml/s during expiration. The pressure measured in the tracheal tube was -0.85 during inspiration, and +0.39 cm H2O during expiration; the end-expiratory pressure in the tube was zero. In two experiments with anaesthetised rats lasting 4-5 hours, ventilation, oxygen uptake, carbon dioxide release and the respiratory exchange ratio were 638 ml/min/kg, 21.7 ml O2(STPD)/min/kg, 16.6 ml CO2(STPD)/min/kg, and 0.77, respectively. There was no significant change in any parameter during the experiment. The micro-valve increases the dead space by approximately 35%, but this is well tolerated by the rats, which compensate by increasing their tidal volume by about 10 to 15%. The major advantage of using the micro-valve in comparison with other methods is the fact that the true difference between inspiratory and mean mixed expiratory gas can be measured with great accuracy. The micro-valve can readily be adjusted for optimal use with a range of animals.