The enzymatic hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA) by phospholipase D (PLD) at the air-water interface was investigated with epifluorescence microscopy. In the PC-PA mixed monolayer doped with octadecyl rhodamine B, the gel phase of PA was observed as dark domains of 2-5 mum diameter at a surface pressure above 25 mN m-1. When PC was spread over a subphase containing Streptomyces chromofuscus PLD, the assemblies of the circular dark domains whose diameter was 2-5 mum appeared at 25 mN m-1. In the case of peanut PLD, few dark domains were observed and their diameter was less than 1 mum. The observed morphological difference in the assemblies of the hydrolysis product by peanut and Streptomyces PLDs indicates that the two PLDs act differently on the monolayer, as has been suggested in the hydrolysis of the monolayer by these PLDs at the oil-water interface. The difference in the hydrolytic action of Streptomyces and plant PLDs suggests that the segregation of the product, PA, from the reaction system as a solid phase is important in efficient hydrolysis.