Abstract Superoxide anion (O – 2 ) and nitric oxide (NO) generation in Dactylopius coccus hemolymph obtained by perfusion and activated with zymosan was studied. Activated hemolymph reduced 3‐[4,5 dimethylthiazolil‐2]‐2,5‐diphenyl tetrazolium bromide. This reduction was prevented by superoxide dismutase (SOD) indicating O – 2 generation. This activity was dependent on temperature, and hemolymph incubated at 75°C lost its activity. Chromatocytes incubated with zymosan released their content and produced O – 2 . Activated hemolymph also produced NO and this activity was prevented in the presence of NG‐nitro‐L‐arginine methyl ester, suggesting that nitric oxide synthase (NOS) might be present in D. coccus hemolymph. The probable source of O – 2 in the D. coccus hemolymph is the anthraquinone oxidation, since commercial carminic dye produced O – 2 during its oxidation by Agaricus bisporus tyrosinase. Gram+ Micrococcus luteus exposed to activated hemolymph were killed in vitro, and addition of NG‐nitro‐L‐arginine methyl ester and D‐Mannitol (a hydroxyl radical scavenger) prevented their killing. The cytotoxic effect produced by the activated hemolymph was not observed with the Gram– bacteria Serratia marcescens . These results suggest that D. coccus activated hemolymph generates reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) that may limit M. luteus growth. Arch. Insect Biochem. Physiol. 65:20–28, 2007. © 2007 Wiley‐Liss, Inc.