Abstract: Melatonin and N‐acetylserotonin (NAS) have antioxidant properties. In the present study, we examined whether melatonin, NAS, and N‐acetyldopamine (NAD) have a modulatory effect on tumor necrosis factor‐α (TNF‐α) synthesis and superoxide production. Differentiated THP‐1‐derived human monocytes were coincubated with Escherichia coli lipopolysaccharide (LPS) and rising concentrations of melatonin, NAS, or NAD. After 24 h, TNF‐α was measured in cell supernatants. In addition, the production of superoxide by HL‐60‐derived human neutrophils upon stimulation with 4‐β‐phorbol 12‐β‐myristate 13‐α‐acetate (PMA) or N‐formyl methionyl‐leucyl‐phenylalanine (fMLP) and increasing concentrations of melatonin, NAS, or NAD was determined. Incubation of THP‐1‐derived monocytes with increasing concentrations of melatonin, NAS, or NAD resulted in a marked decrease in LPS‐stimulated TNF‐α production, which was dose‐dependent and on the order of 96‐98%. Incubation of HL‐60‐derived neutrophils with increasing concentrations of melatonin, NAS, or NAD resulted in a modest decrease in PMA‐stimulated superoxide production, which was dose‐dependent. At the 100 μM dose, melatonin, NAS, or NAD resulted in a 14 ± 4%, 30 ± 1%, and 29 ± 1% decrease in PMA‐stimulated superoxide production, respectively. Coincubation of HL‐60 cells with melatonin, NAS, or NAD also resulted in a modest dose‐dependent decrease in fMLP‐stimulated superoxide production. At the 100 μM dose, melatonin, NAS, or NAD resulted in a 13 ± 1%, 14 ± 1%, and 14 ± 1% decrease in superoxide production, respectively. Our results indicate that the inhibitory effect of melatonin, NAS, or NAD on LPS‐induced TNF‐α production is robust and dose‐dependent. These compounds are equally effective in attenuating the generation of oxidant radicals, although to a lesser degree.