Microsomes (100,000 g pellet containing mixed membrane fractions but primarily endoplasmic reticulum) were isolated from shoots of corn, shattercane, and woolly cupgrass grown from naphthalic anhydride treated or untreated seed to determine if metabolism of bentazon, nicosulfuron, and primisulfuron could be demonstrated in the preparations. Corn is tolerant of all three herbicides, shattercane is tolerant of bentazon, and woolly cupgrass is tolerant of bentazon and primisulfuron. Naphthalic anhydride treatment was required for detectable bentazon, nicosulfuron, and primisulfuron hydroxylation in corn microsomes and for bentazon hydroxylation in woolly cupgrass microsomes. Bentazon hydroxylation was low, but detectable, in microsomes from shattercane shoots without naphthalic anhydride treatment. Naphthalic anhydride-treated corn microsomes hydroxylated 292, 120, and 52 pmol mg−1 protein min−1 of bentazon, nicosulfuron, and primisulfuron, respectively. Primisulfuron (19 pmol mg−1 protein min−1), but not nicosulfuron, was hydroxylated in woolly cupgrass microsomes. Neither nicosulfuron nor primisulfuron was hydroxylated in shattercane microsomes. Bentazon and primisulfuron inhibited nicosulfuron hydroxylation in corn microsomes. Bentazon, but not nicosulfuron, also inhibited primisulfuron hydroxylation in the corn microsomes. This indicates that the three herbicides can interact at the same cytochrome P-450(s) in corn. Primisulfuron hydroxylation was not inhibited by either bentazon or nicosulfuron in woolly cupgrass microsomes. This suggests that the cytochrome P-450(s) for primisulfuron hydroxylation are different between corn and woolly cupgrass. Also, bentazon hydroxylation in corn and shattercane microsomes was inhibited by the cytochrome P-450 inhibitor tetcyclasis, while that in woolly cupgrass was not. Again, this suggests a difference in the cytochrome P-450(s) responsible for bentazon metabolism among the species. Although absolute conclusions comparing in vitro microsomal activities to whole plant herbicide tolerance cannot be made because it is unknown whether the same cytochrome P-450(s) are studied in microsomes from naphthalic anhydride-treated tissue as are responsible for in vivo herbicide metabolism, there was a broad correlation between metabolism of a particular herbicide in microsomes of a species and the species' tolerance of that herbicide.