To determine biochemical changes associated with early parasite development, Haemonchus contortus larvae were cultured in vitro to the fourth stage (L4). Infective larvae developed from third to fourth stage in 48-96 h. Metabolic activity increased following stimulus of infective stages by CO2 secretion/excretion of significant amounts of protein into cultures and larval feeding did not occur until larvae had molted to the fourth stage. Larval feeding, as monitored by the ability of larvae to ingest fluorescein-labeled albumin, correlated with molting to the fourth stage and only fourth stage larvae were observed to feed. Fourth stage larvae secreted/excreted several enzymes into culture media including a metalloprotease, an acid phosphohydrolase, a cathepsin C-like enzyme, a phospholipase C-like enzyme and an N-acetyl-beta-D-glucosaminidase. Excretory-secretory (ES) products produced by L4 had antigenic homologies with parasite products produced during the second molt and with proteins and glycoproteins extracted from third and fourth stage larvae. ES products were recognized by sera from sheep infected with H. contortus. The enzymes identified here serve as markers for maturation to the fourth larval stage as well as the initiation of feeding and are likely to be involved in extracorporeal digestion. Further, they might serve as potential targets for immune or chemical control of trichostrongyle infections.