The hypothesis of this work is that the 'serotonin' or 5-hydroxytryptamine (5-HT)(1A) receptor, which activates the extracellular signal-regulated kinase (ERK) through a G(i)betagamma-mediated pathway, does so through the intermediate actions of reactive oxygen species (ROS). Five criteria were shown to support a key role for ROS in the activation of ERK by the 5-HT(1A) receptor. (1) Antioxidants inhibit activation of ERK by 5-HT. (2) Application of cysteine-reactive oxidant molecules activates ERK. (3) The 5-HT(1A) receptor alters cellular redox properties, and generates both superoxide and hydrogen peroxide. (4) A specific ROS-producing enzyme [NAD(P)H oxidase] is involved in the activation of ERK. (5) There is specificity both in the effects of various chemical oxidizers, and in the putative location of the ROS in the ERK activation pathway. We propose that NAD(P)H oxidase is located in the ERK activation pathway stimulated by the transfected 5-HT(1A) receptor in Chinese hamster ovary (CHO) cells downstream of G(i)betagamma subunits and upstream of or at the level of the non-receptor tyrosine kinase, Src. Moreover, these experiments provide confirmation that the transfected human 5-HT(1A) receptor induces the production of ROS (superoxide and hydrogen peroxide) in CHO cells, and support the possibility that an NAD(P)H oxidase-like enzyme might be involved in the 5-HT-mediated generation of both superoxide and hydrogen peroxide.