Two successive factorial designs followed by two response surface methodology were applied to optimize protopectinase production by Geotrichum klebahnii. Factorial designs were used to study the effect of 11 variables (mineral pool and pH) on enzyme production. Only pH and Fe2+ had significant effect on the protopectinase production in the conditions of the assay, the interaction between them not being significant. According to this result pH and Fe2+ were multivariate according to a Doelhert design. The central points were pH 3.5 and Fe2+ 1.8 mu mol L-1. The results show a negative effect of pH and a positive (quadratic) effect of Fe 21 on enzyme production. The second Doelhert design was centered at pH 3.3. A relative maximum was obtained at pH 2.8 and Fe2+ 0.540 mu mol L-1, where the enzyme activity obtained was 236 U/mL. This value is two times higher than the value reported elsewhere. (c) 2006 Elsevier Ltd. All rights reserved.