In vitro culture methods were utilized to investigate specific nutritional requirements of Brugia pahangi larvae. Infective third-stage larvae (L3) isolated from Aedes aegypti (Liverpool) mosquitoes were cultured in NCTC 135:IMDM (NI) medium plus various types of serum under 5% CO2/air and 5% CO2/N2 atmospheres. Larvae grew, developed, and molted to the fourth stage (L4); however, further growth and differentiation beyond the mid-L4 were minimal. Serum supplementation was necessary to induce molting of B. pahangi L3s. Iron-supplemented calf serum (FeCS) consistently promoted higher percentages of molting than the other 4 types of serum tested. Specific nutritional requirements for B. pahangi larvae were assessed through testing of supplements known to be important for growth of mammalian cells or other parasites in vitro. Addition of various concentrations and combinations of hemoglobin, insulin, transferrin, selenium, albumin, cholesterol, or a cell feeder layer did not improve larval growth, molting, or survival compared to that obtained with FeCS alone. The L4s derived from in vitro culture, when injected intraperitoneally into jirds, developed to sexually mature, microfilarial-producing adults in a normal in vivo time frame. Developing L4s isolated from jirds molted to young adults in vitro in NI medium plus serum.