Mammalian flavin-containing monooxygenases are known to oxygenate prochiral sulfides often with a high degree of stereoselectivity. In the present study we used two homologous series of n-alkyl aryl sulfides, Ar-S-(CH 2 ) n H, (where Ar=p-tolyl or 2-naphthyl and n=1-7), to probe the active site topography of rabbit lung FMO2. Purified FMO2 was incubated with each member of the the p-tolyl or 2-naphthyl series and the stereochemistry of NADPH-dependent enzymatic sulfoxidation was determined. Within the p-tolyl series each member was a substrate for FMO2 and a complete reversal of stereospecificity was observed as the n-alkyl chain length increased from the methyl homolog [99% (R)] to the heptyl derivative [97% (S)]. In contrast, FMO2 generated metabolites from only the short-chain 2-naphthyl sulfides and in each case only the (R)-sulfoxides were formed. The data are discussed in terms of an active-site model for FMO2 which contains two binding determinants.