A population of early to late third-stage larvae (L3) of Ascaris suum obtained from the lungs of swine 7 days after infection developed to the fourth stage (L4) in stationary, multi-well, culture plates in an atmosphere of 5% CO2 in air. Larval survival, growth and morphogenesis were evaluated in five culture systems consisting of Dulbecco's Modified Eagles' Medium alone (DM) or containing a serum supplement (DM-S) or a tripeptide (1-glycyl-1-histidyl-1-lysine) at final concentrations of 20, 200, and 1,000 ng/ml (respectively, DM-20, DM-200, and DM-1,000). The rate of development and morphogenesis of larvae from L3 to L4 was optimal in the DM-S culture system and similar to that observed in vivo. However, development beyond L4 was retarded in vitro; sex was not distinguished until 21 days in culture (DIC) and the largest L4 obtained after 52 DIC was 9.2 mm long. Although larval growth and development were similar in all systems tested through 14 days in culture, higher yields of advanced stages of L4 were obtained in systems DM-200 and DM-S. Developing larvae released metabolic products into the culture media that stimulated a specific blastogenic response in lymphocytes obtained from A. suum-infected swine.