Sexual development in the mated cultures of Dictyostelium discoideum is initiated by one or more pheromones. In agar plate cultures of NC4, the presence of the pheromone becomes detectable by 22 h and reaches its maximal activity by 24 h. Cells of V12 from plate cultures also reach their maximal responsiveness at 24 h. In liquid culture, the NC4 pheromonal activity is detectable by 10 h and increases to a maximum at 15 h before declining so that by 30 h no activity is detectable. The V12 cells develop the competence to respond to the pheromone by 10 h and increase to a peak responsiveness by 15 h after which they lose the ability to respond. These data support earlier experiments demonstrating that sexual pheromones exist in the cellular slime moulds. The results of the liquid culture experiments in which the pheromonal induction of macrocyst formation reaches levels equivalent to 30% of mated cultures (NC4 × V12) also suggest that this pheromone bioassay system is sensitive enough to permit the isolation and characterization of the sexual pheromones of D. discoideum.