The ability of six fungi to solubilize natural-occurring gypsum was tested in vitro. The solubilization process was monitored by the production of a clear zone (halo) around or underneath the growing colony on Czapek–Dox agar containing 0·5% (w/v) gypsum (CaSO4.2H2O). Aspergillus niger, Penicillium bilaii, P. simplicissimum and Paxillus involutus displayed differential solubilization activities depending on the supplemented nitrogen source. Colonies grown on nitrate-containing medium showed the ability to solubilize gypsum, but when ammonium (at equivalent nitrogen) was used there was a significant reduction in solubilization. It was found that liquid cultures of nitrate-grown fungi produced substantial amounts of oxalic acid, whereas in ammonium-containing medium oxalic acid was only detected in small amounts. The production of citric and gluconic acid under these experimental conditions was low in both media, although the involvement of citric acid in gypsum solubilization is possible. Coriolus versicolor and Phanaerochaete chrysosporium did not exhibit any solubilization activity in nitrate- or ammonium-containing medium. Additionally these two fungi excreted small quantities of oxalic acid in both media with no citric acid being produced in liquid medium. Concomitant with the solubilization process and inside the clear solubilized zone, A. niger, Pax. involutus and P. bilaii produced crystals of differing shapes and abundance depending on the fungal strain. No crystals were produced by P. simplicissimum. These crystals were identified as calcium oxalate based on HPLC analysis and energy-dispersive X-ray microanalysis. Abundance of these crystals was found to be correlated with both oxalic acid production and the acidity of the medium. It is concluded that gypsum solubilization was predominantly achieved by both oxalic acid, which was accompanied by formation of calcium oxalate crystals, and citric acid production rather than the acidity of the medium. The results are discussed in relation to the significance of such an activity in agricultural applications, e.g. land reclamation, as well as the possible roles played by these fungi in mineral cycling.