A multiwell culture system was used to assay the effects of 12 known anthelmintic compounds on Ascaris suum larval development from 2nd-stage (L2; hatched from eggs) to early 3rd-stage (L3) and from in vivo-derived late L3 to early 4th-stage (L4). Larval survival, development, and motility were monitored for drug effects. Development of L2 to L3 was sensitive to thiabendazole, albendazole (ABZ), ABZ/sulfoxide, ABZ/sulfone (SO), mebendazole, L-tetramisole, D-tetramisole, piperazine, or closantel at a concentration of 0.01 microgram/ml; however, the effects of these drugs on larval development did not correlate well with known effects in vivo. The development of L3 to L4 was blocked by ABZ or mebendazole at 0.01 microgram/ml, by thiabendazole or ABZ/sulfoxide at 0.1 microgram/ml, and by ABZ/SO at 1.0 microgram/ml; however, except for ABZ/SO, most larvae were viable at these concentrations. In contrast, L-tetramisole or morantel appeared to inhibit development of L3 to L4 and to reduce survival at concentrations of greater than or equal to 1 microgram/ml; however, D-tetramisole was at least 10 times less effective. Haloxon, ivermectin, and closantel blocked development of L3 to L4 at 0.1, 1, and 10 micrograms/ml, respectively, in the absence of serum, but their activity was reduced by the presence of serum. Seemingly, in vitro development of A suum larvae was a convenient and sensitive bioassay for anthelmintic activity and could serve as a screen for anthelmintic residues in edible tissues.