The establishment and optimization of in vitro primmorph formation from a Chinese sponge, Stylotella agminata (Ridley), collected from the South China Sea, were investigated. Our aims were to identify the key factors affecting primmorph formation in this species and to optimize the technique for developing an in vitro primmorph culture system. The size of dissociated cells from S. agminata is relatively small, in the range between 5 and 10 microm. Round-shaped primmorphs of less than 100 microm were formed 3 days after transferring the dissociated cells into seawater containing Ca(2+) and Mg(2+). The effect of various cell dissociation conditions, inoculum cell density, concentration of antibiotics, pH, and temperature was further investigated upon the formation of primmorphs. The time required for primmorph formation, primmorph size distribution, and the proliferating capability were microscopically documented. Healthy sponge S. agminata, inoculum cell density and culture temperature play a critical role for the successful formation of primmorphs and that the microbial contamination will have to be controlled.