Concerning the antibacterial test in vitro, one of the most important factors will be the solubility of compounds in water. Such substances as gramicidin J₁ and chloramphenicol can be tested in a usual method owing to its strong activity though their solubility is quite slight, but the activities of the compounds low in both activity and solubility can not be determined with accuracy in usual manner. As such compounds are usually of no practical use, there is no inconvenience practically, but when one attempts the study of such basic problem as the relationship between chemical structures and antibacterial activities, the low solubility becomes a great interference. The authors met with this during the investigation of the acyl derivatives of gramicidin J₁, which are highly insoluble in water. In order to solubilize these materials, we tried to use surface active agents, and in reality their utilization for this purpose has been reported in many cases¹⁾. It goes without saying that this method is only adoptable when the agents have no influence upon the activities of substances, but the reports of many researchers do not always show complete agreement, since some say it has no influence²⁾, some claim it lowers the activities^3,4) and others describe it strengthens them^5,6). Then we reinvestigated this problem in hopes of finding appropriate agents applicable mainly to the gramicidin derivatives.