The toxicity of the organophosphorus poison soman (pinacolylmethylphosphonofluoridate) is attributable to its irreversible inhibition of the enzyme acetylcholinesterase. In addition, soman binds irreversibly to a number of noncholinesterase tissue binding sites which appear to be its major means of in vivo detoxification. This study was conducted to determine the hepatic subcellular localization of these sites. Subcellular fractions of liver from male Sprague-Dawley rats (200-250 g) were prepared by differential and isopycnic density gradient centrifugation. The binding of [14C]soman to these subcellular fractions was determined in the presence and absence of cresylbenzodioxaphosphorin oxide (CBDP), a compound that binds irreversibly to the noncholinesterase soman binding sites. Crude fractionation of liver homogenates into nuclear, mitochondrial, microsomal, and soluble fractions revealed that 78% of the total CBDP-sensitive binding activity was localized in the nuclear and microsomal fractions. Further purification of these fractions indicated that all of the homogenate binding activity could be accounted for in the purified microsomal fraction. When purified liver microsomes were solubilized and fractionated on linear sucrose gradients, 90% of the CBDP-sensitive soman binding activity cosedimented with carboxylesterase activity which suggests that these binding sites are carboxylesterase.