We studied the effect of CO(2) on the in vitro cultivation of Anisakis simplex, an aquatic parasitic nematode of cetaceans (final hosts) and fish, squid, crustaceans and other invertebrates (intermediate/paratenic hosts), and, occasionally, of man (accidental host). The results showed that a high pCO(2), at a suitable temperature, is vital for the optimum development of these nematodes, at least from the third larval stage (L3) to adult. After 30 days cultivation in air, molting to L4 (fourth larval stage) was reduced to 1/3, while survival was about 1/3 of that when cultivated in air + 5% CO(2). The activity of the CO(2)-fixing enzymes, PEPCK and PEPC, was also studied. Throughout the development of the worms studied, PEPCK activity was much higher than that of PEPC (e.g., 305 vs. 6.8 nmol/min.mg protein, respectively, in L3 collected from the host fish). The activity of these enzymes in the worms cultivated in air + 5% CO(2) was highest during M3, and was also generally higher than that of those cultivated in air only, especially during molting from L3 to L4 (e.g., in recently molted L4, PEPCK activity was 3.7 times greater than that of PEPC 2.9 times greater than when cultivated in air).