At the onset of development, the single cells of the eukaryotic micro-organism Dictyostelium discoideum secrete proteolytic activity which can be assayed using the insoluble substrate remazolbrilliant blue hide. The activity is not secreted by exponentially growing cells, but does appear extracellularly at the onset of the stationary growth phase. When growth phase cells are resuspended in non-nutrient buffer, proteolytic activity begins to appear outside the cells. It accumulates in the buffer at a rate similar to that observed for 2 glycosidases of lysosomal origin and reaches a maximum after about 2 h of incubation. After 3--4 h incubation, centrifugation of the non-nutrient buffer removes the cells, producing a supernatant which we refer to as conditioned medium. Subsequent experiments with conditioned medium showed: (a) its incubation with purified plasma membranes results in the release of polypeptides which can be recovered and, when displayed on polyacrylamide gels, can be shown to be stage specific; and (b) that conditioned medium can decrease the rate of detachment of cells from a collagen substratum. Both effects can be prevented by the addition of remazolbrilliant blue hide suggesting that they are due to proteolytic activity present in the conditioned medium. Finally, we were able to show that conditioned medium contains components which, when spread over the bottom of plastic Petri dishes, enhance the rate of multicellular structure formation. Additional studies showed that this effect of conditioned medium could also be brought about by components which remained behind on uncoated plastic dishes after the removal of a D. discoideum cell layer. These data may be accommodated to a model in which the protease secreted during the onset of development acts on the cell membrane releasing components which coat the substratum and facilitate migration and multicellular structure formation.