Electrophoretically mediated microanalysis (EMMA) was applied for the study of kinetic parameters of the bisubstrate enzymatic reaction of rhodanese. The Michaelis constants (K-m) for both substrates and the effect of temperature on rhodanese reaction were evaluated by means of the combination of the EMMA methodology with a partial filling technique. In this setup, the part of the capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. The enzymatic reaction was performed in 25 mM N-(2-hydroxymethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) buffer (pH 8.5) while the low pH background electrolyte 100 mm beta-alanine-HCl (pH 3.5) was used for separation of substrates and products that are the inorganic anions. The estimated value of K-m for thiosulfate of 1.30 x 10(-2) m was consistent with previously published values; the K-m for cyanide of 7.6 x 10(-3) m was determined for the first time. In addition, the type of kinetic mechanism of enzymatic reaction was also elucidated. The finding of the double displacement (ping-pong) mechanism is in accordance with previous literature data. Also, the experimentally determined temperature optimum of the rhodanese-catalyzed reaction around 20-25degreesC agreed with literature values.