Culture conditions suitable for production of PDM phosphatase by the fungus, Fusarium moniliforme, were investigated and established as follows. The medium contained 3% (w/v) glucose, 0.1% (w/v) ammonium sulfate and 0.1% (w/v) yeast extract in 50 mM sodium citrate buffer (pH 5.8) and the temperature was 25℃. In an early stage under these conditions, there was production of another unstable phosphatase presumably specific for phosphomonoesters. After the decline of this phosphatase activity at about 40 h of the culture time, PDM phosphatase began to increase and reached a plateau at about 100 h. This time course of production was slightly different from that observed under the same conditions for ribonuclease F1, another phosphoric acid hydrolyzing enzyme secreted by the fungus.