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 Fe 2+ 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 Fe 2+ were multivariate according to a Doelhert design. The central points were pH 3.5 and Fe 2+ 1.8μmolL −1 . The results show a negative effect of pH and a positive (quadratic) effect of Fe 2+ on enzyme production. The second Doelhert design was centered at pH 3.3. A relative maximum was obtained at pH 2.8 and Fe 2+ 0.540μmolL −1 , where the enzyme activity obtained was 236U/mL. This value is two times higher than the value reported elsewhere.