The effects of subchronic oral administration of metrifonate, a long-acting cholinesterase (ChE) inhibitor, on cholinergic neurotransmission were assessed in young adult male Wistar rats. Animals were treated twice daily with metrifonate. In a pilot study testing a 100 mg/kg dose of metrifonate for up to 14 days, ChE activity was found to steadily decrease to reach maximum inhibition levels of about 55%, 80% and 35% in brain, erythrocytes and plasma. Steady-state inhibition levels were attained by the 10th day of treatment. When metrifonate-treatment was discontinued, ChE activity in plasma returned to control levels within another day, while erythrocyte and brain ChE activity took more than 2 weeks to recover. In subsequent dose-response studies, metrifonate treatment was given for 3 and 4.5 weeks at doses of 0, 12.5, 25, 50, and 100 mg/kg, to different groups of animals, respectively. Correlation analysis indicted that brain ChE inhibition was more accurately reflected by erythrocyte than by plasma ChE inhibition, although all effects were highly correlated. The changes in ChE activity were not paralleled by changes in other parameters of the cholinergic neurotransmission, such as acetylcholine synthesis rate or acetylcholine receptor binding. It is therefore concluded that repeated administration of metrifonate to rats induces a long-lasting inhibition of ChE activity in a dose-related and predictable manner, which is neither subject to desensitization nor paralleled by counterregulatory downregulation of muscarinic or nicotinic receptor binding sites in brain.