The phospholipase D (PLD)-encoding gene of Streptomyces antibioticus fused with the pectate lyase B (PelB) signal sequence was expressed in recombinant Escherichia coli under the control of the T7 lac promoter, Most of the PLD activity was detected in the culture supernatant. The N-terminal amino acid sequence of the recombinant PLD was identical to that of the wild-type PLD, suggesting that the processing of the PelB signal peptide occurred correctly and that the PLD produced by E. coil was identical to the wild-type enzyme. The production of PLD was improved by genetic and fermentation techniques. About 3 mg of PLD/l medium was obtained under optimal conditions, comparable to the amount of PLD produced by S. antibioticus. The results of analysis of the distributions of intracellular marker enzymes suggested that neither cell lysis nor periplasmic disruption was the cause of the secretion of PLD.