The mechanisms of action of mineral phosphate solubilization (MPS) were studied in the wild-type Mps⁺Penicillium rugulosum strain IR94-MF1 and in negative (Mps⁻) and superpositive (Mps⁺⁺) mutants derived from it. MPS activities were measured in liquid media using sucrose as C source, four N (arginine, nitrate, nitrate+ammonium and ammonium) and P sources (KH₂PO₄, hydroxyapatite, FePO₄ and AlPO₄). Ammonium significantly (P<0.01) decreased phosphate solubilization, and this activity was 1–66 times higher in the Mps⁺⁺ mutant than in the wild-type depending on the P and N sources used. The Mps⁺ phenotype was strongly associated with the production of gluconic or citric acids. The results also suggest for the MPS⁻ mutant the involvement of the H⁺ pump mechanism in the solubilization of small amounts of phosphates.