Nuclear factor kappaB (NF-kappaB) is an important stress-induced transcription factor in many cell types. In this study we examined if the hepatic tumor promoting agent phenobarbital could activate NF-kappaB in the liver. Rats were fed 0.05% phenobarbital in the diet for 3, 6, or 10 days. Benzyloxyresorufin dealkylase activity was greatly increased in rats receiving phenobarbital. The DNA binding activity of NF-kappaB was determined using electrophoretic mobility shift assays. After 3 days, NF-kappaB activity was increased two-fold in rats receiving phenobarbital. NF-kappaB activity continued to increase at 6 and 10 days in rats fed phenobarbital, with a four-fold increase seen at day 10. Cold competition and antibody supershift experiments showed the DNA binding activity to be specific for NF-kappaB. These data show that phenobarbital activates hepatic NF-kappaB in rats.