Three secreted acid phosphatases had previously been characterized from Aspergillus ficuum grown under conditions of limited phosphate. One of these could not be readily separated from AFPhyB, a pH 2.5 optimum acid phosphatase with phytase activity. From extensive protein sequence analysis and subsequent cloning of the gene, we have shown that the AFPhyB protein fraction contains a fourth secreted acid phosphatase (AFPhoA) that has 64% homology to a phosphate-repressible acid phosphatase from Penicillium chrysogenum. Garnier plot analysis revealed that the putative phosphate catalytic domain of AFPhoA at His215Asp216 is similar to those of other acid phosphatases, but that AFPhoA lacks the phosphate-binding motif RHGXRXP of known histidine phosphatases.