Pectic substances occur in superior plants and are constituted by a main chain of polygalacturonic acid branched with other sugars. These substances are degraded by extracellular pectolytic enzymes mostly produced by filamentous fungi. These enzymes may be classified in esterases (pectin esterase) and depolymerases (polygalacturonases and lyases) and they have a high potential for biotechnological use in olive oil extraction, oils recovery from fruit peel, fruit juices clarification, wine production and textile industry. The aim of this work was to select fungi with good production of pectinases, from soil or plants of several São Paulo areas, according to Biota program. In these isolates we optimized the cultivation conditions in terms of nutritional sources and physicochemical parameters. Preliminarily, the selected fungi were Penicillium herquei, Aspergillus caespitosus and Rhizopus microsporus var. rhizopodiformis, which produced high levels of polygalacturonase in Vogel, SR and Adams media, respectively. All the media were supplemented with 1% pectin (Sigma) as carbon source. Cultures were carried out under agitation, at 35-40ºC. Penicillium herquei and A. caespitosus exhibited the best production of polygalacturonase after 48h, while for R. microsporus var. rhizopodiformis it was after 72h. Regardless the longer culture time required for optimal polygalacturonase production, R. microsporus var. rhizopodiformis was the best enzyme producer. Therefore, the study was focused on this microorganism. Interestingly among 32 carbon sources tested, lemon peel was the best inductor of pectinolytic enzymes (polygalacturonases, pectate lyase and pectin lyase). For the polygalacturonase produced by R. microsporus var. rhizopodiformis, it was observed a maximum enzymatic activity at 65ºC and pH 3.5. The enzyme was stable up to 60ºC. The polygalacturonase activity was inactivated 28%, 39% and 52% at 60, 65 and 70ºC, respectively, after 90 minutes of assay.