Penicillium cyclopium triacylglycerol lipase production was maximized in stationary batch culture. We used a surface response methodology based on a Doehlert experimental design to study the effect on the lipase activity released in the culture medium of the three most important factors: substrate concentration, pH and inoculum. Besides reducing the number of experiments required for optimization, this technique allowed us to quantify the lipase activity in any part of the experimental domain. We determined an optimal set of conditions for high lipase production: 1% substrate (corn steep), pH 5.5 and an inoculum of 10(4) spores/ml. Between conditions giving the minimum and the maximum lipase production, we observed a nine-fold increase of both the predicted and measured values.