Rhizomucor miehei lipase, Lipozyme IM-20, was used to catalyze the esterification reaction between isobutyric acid and isoamyl alcohol to synthesize isoamyl isobutyrate in n-hexane. Response surface methodology (RSM) based on a five-level, four-variable central composite rotatable design (CCRD) was employed for optimization with respect to four important reaction variables enzyme/substrate (E/S) ratio, substrate concentration, reaction time, and temperature. Conversion was efficient in an acid concentration range of 0.5-2.5 M, even when a very low E/S ratio (5 g/mol) was employed. Although an increase in temperature resulted in improved reaction rates, overall conversions remained practically unchanged, as the yields obtained at lower temperatures (30-40 degreesC) were higher. Reaction time also exhibited a less significant effect on ester yields at higher E/S ratios. The optimum conditions predicted for achieving maximum ester yield (2.2 M) are: E/S ratio, 19.6 g/mol; substrate concentration, 2.5 M; reaction time, 18 h and temperature, 26.5 degreesC.