Cultures of Carcinoscorpius rotundicauda amoebocytes in Grace's insect (modified) medium supplemented with 20% foetal bovine serum, were stimulated with pokeweed mitogen at 0.5 to 50 micrograms/ml for 24 to 72 h at 26 degrees C. DNA synthesis was monitored by measuring the uptake of 3H-thymidine. The optimal concentration of pokeweed mitogen was established at 2.5 micrograms/ml. The ideal duration of stimulation was 48 h. Under these conditions, amoebocyte cultures showed increases in uptake of 3H-thymidine up to 55-fold. Attempts at stimulation of these amoebocytes with phytohaemagglutinin, concanavalin A and endotoxin showed no increase in 3H-thymidine uptake. Nevertheless, positive stimulation by pokeweed mitogen demonstrates the potential of such an approach to establishing a permanent amoebocyte cell line for lysate production. This stimulation was reversed by 5 mM hydroxyurea, suggesting a true induction of DNA synthesis.