Mycobiont isolation experiments were performed on six species of Ramalina from Brazil: R. celastri, R. complanata, R. dendriscoides, R. gracilis, R. peruviana and R. sprengelii. This study aimed to optimize the culture conditions and nutrient requirements of the selected mycobionts. The aposymbiotic R. complanta was successfully isolated from ascospores, while aposymbiotic R. peruviana was obtained from thallus fragments. In R. peruviana the production of secondary metabolites was investigated under aposymbiotical growth conditions using HPLC. When cultivated on solid medium, this mycobiont produced the typical chemosyndrome (sekikaic acid and satellite compounds), found in the voucher lichen thallus. When cultivated in liquid medium (immersed in malt yeast medium in the absence of agar), only one, the major lichen substance, sekikaic acid, was synthesized by the fungus. In addition, atranorin was formed, but was not detected in any of the voucher specimens. Red pigments were found in solid and liquid cultures. These were separated into two compounds, but could not be fully identified. R. celastri spores germinated, but did not form mycelia. R. dendriscoides, R. gracilis and R. sprengelii were not successfully cultivated in aposymbiotic conditions, although eight different culture media were tested.