A new semi-quantitative agar plate test for the detection of extracellular rhamnolipids has been developed. These biological anionic tensides (biosurfactants) form an insoluble ion pair with the cationic tenside cetyltrimethylammonium bromide and the basic dye methylene blue which was included in mineral agar plates. On the light blue agar, productive colonies ofPseudomonas spec. were surrounded by dark blue halos. The test is specific for anionic biosurfactants and can be applied to other glycolipid producing microorganims.