Several cell lines from the pupae of the noctuid moth species Spodoptera frugiperda, Heliothis zea, and Trichoplusia ni were isolated on a synthetic medium containing insect hemolymph and turkey serum. These lines were progressively adapted to improved media free of insect hemolymph but containing one or more of the following sera: turkey, chicken, and fetal calf. Primary culture tissue disruption was improved by substituting collagenase for trypsin. Primary culture survival was improved by controlling the total tissue volume per unit medium volum, and by the addition of glutathione to prevent melanization and to improve cell adherence to the substrate. Culture servival was also improved by heat treatment of sera, control of medium osmolality, and changes in the basal medium and serum supplementation. Some of these changes also resulted in improved growth giving higher maximal cell counts. Comparative cell growth on the various media was graphed and generation times given.