Advanced third-stage larvae of Gnathostoma spinigerum were cultured in RPMI-1640, with various supplements at 37 C under 5% CO2 in air for 300 days. The most suitable medium supplement for worm development was 10% fetal calf serum, 1% dog serum, and 0.25% dog hemolysate. After approximately 180 days of cultivation, some larvae molted to the fourth stage as distinguished by 8 transverse rows of cephalic hooklets and well differentiated sex organs. The maximum body length and width of these larvae were 18.6 mm and 1.1 mm, respectively. Six of 50 larvae (12%) developed to the fourth stage, with a 32% survival rate at the end of cultivation. Although the highest survival rate (70%) of the worms was observed in the medium supplemented with 25 mM NaHCO3, only 4% developed into fourth stage larvae. The addition of fetal calf serum, dog serum, and dog hemolysate was essential for growth and development.