Inhibition of rat liver microsomal carboxylesterase (CEase) by O-ethyl O-p-nitrophenyl phenylphosphonothioate (EPN) and binding of EPN oxygen analog to microsomal CEase were enhanced by addition of NAD or NADP. This was more prominent in addition of NAD than NADP. No potentiation of anti-CEase action of EPN by NAD was seen when pure esterase (E.C. 3.1.1.1) instead of liver microsomes was used as an enzyme source. This effect of NAD in microsomal CEase was significantly decreased when N-ethylmaleimide or p-chloromercuribenzoic acid was added. From these findings, it is strongly suggested that NAD-mediated potentiation of the anti-CEase action of EPN might be attributed to the increase in formation of NADH from NAD by microsomal dehydrogenase(s) containing a sulfhydryl group, leading to a subsequent increase in formation of the EPN oxygen analog from EPN, and in turn, CEase inhibition was enhanced.