The dark respiration rates of primary leaf mesophyll protoplasts of barley (Hordeum vulgare L. cv. Patty) were measured by a Clark-type oxygen electrode and Cartesian-diver microrespirometry, in order to compare the two techniques. Factors found to be important in determinations of respiratory rates of protoplasts were the numbers of protoplasts per unit volume of suspension, the age of protoplast preparations and the stirring speed of the magnetic follower when using the oxygen electrode. Increases in numbers of protoplasts per unit volume caused a marked decrease in respiration using both techniques. Below 2000 protoplasts μl−1 both techniques provided rates of dark respiration within the same range until 1000 protoplasts μl−1, below which a marked increase in respiration occurred. Respiratory rates of protoplasts declined by 32 % from initial values over a 30 h period after isolation in the dark. In addition, an increase in the stirring rate in the reaction vessel of the oxygen electrode had the effect of reducing both the viability of protoplasts and their rates of oxygen uptake. It is suggested that consistent standardization of procedures, especially in terms of numbers of protoplasts per unit volume, is necessary for assay techniques for respiration in protoplasts. The advantages and disadvantages of using Cartesian-diver microrespirometry compared to oxygen electrodes are also discussed.