The extent of DNA adduct formation by alachlor [ArN(CH2OCH3)C(O)CH2Cl wherein Ar is 2,6-diethylphenyl] and its metabolites is used as a guide to deduce the causal agent(s) in the carcinogenicity of this major herbicide. [14C-phenyl]Alachlor is compared to its two metabolic cleavage products, [14C-phenyl]2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA) [ArNHC(O)CH2Cl] and [14C-phenyl]2,6-diethylaniline (DEA) (ArNH2), and to [14C-methoxy]alachlor in various in vitro and in vivo systems. Horseradish peroxidase and hydrogen peroxide activate DEA, but not CDEPA or alachlor, for formation of adducts with calf thymus DNA, which probably involves 2,6-diethylnitrosobenzene (ArNO) as an intermediate. Mouse liver microsomes and NADPH are both required to enhance the binding from each labeled preparation to calf thymus DNA; 4-fold higher labeling is observed from [14C-methoxy]- than from [14C-phenyl]alachlor. This 4-fold preferential DNA labeling from the 14C-methoxy compound is likewise found in the liver of mice treated intraperitoneally. Mouse liver protein and hemoglobin are also labeled, in vivo, with [14C-phenyl]alachlor, -CDEPA and -DEA, and, as with the DNA, the labeling of these proteins is 1.5- to 2-fold higher with [14C-methoxy]alachlor. Metabolic studies indicate that ArN(CH2OCH2OH)C(O)CH2Cl is an intermediate in forming CDEPA and presumably formaldehyde in the mouse liver microsomal mixed-function oxidase system and in yielding the O-glucuronide of ArN(CH2OH)C(O)CH2Cl in the urine of alachlor-treated mice. These findings point to the N-CH2OCH2OH metabolite or formaldehyde as a reactive intermediate in forming a DNA-adduct and as a candidate proximate carcinogen.