Selenium has been implicated as a promising chemopreventive agent for prostate cancer. Whereas the anticancer mechanisms have not been clearly defined, one hypothesis relates to selenium metabolites, especially the monomethyl selenium pool, generated under supranutritional selenium supplementation. To explore potential molecular targets for mediating the chemopreventive activity, we contrasted the effects of methylseleninic acid (MSeA), a novel precursor of methylselenol, versus sodium selenite, a representative of the hydrogen selenide metabolite pool, on apoptosis execution, cell cycle distribution, and selected protein kinases in DU145 human prostate cancer cells. Exposure of DU145 cells to 3 microM MSeA led to a profound G1 arrest at 24 h, and exposure to greater concentrations led to not only G1 arrest, but also to DNA fragmentation and caspase-mediated cleavage of poly(ADP-ribose) polymerase (PARP), two biochemical hallmarks of apoptosis. Immunobiot analyses indicated that G1 arrest induced by the subapoptogenic doses of MSeA was associated with increased expression of p27kip1 and p21cip1, but apoptosis was accompanied by dose-dependent decreases of phosphorylation of protein kinase AKT and extracellular signal-regulated kinase (ERK1/2) in the absence of any phosphorylation change in p38 mitogen-activated protein kinase (p38MAPK) and c-Jun NH2-terminal kinase (JNK1/2). In contrast, selenite exposure caused S-phase arrest and caspase-independent apoptotic DNA fragmentation, which were associated with decreased expression of p27kip1 and p21cip1 and increased phosphorylation of AKT, JNK1/2, and p38MAPK. Although apoptosis induction by MSeA exposure was not sensitive to superoxide dismutase added into the cell culture medium, cell detachment and DNA nucleosomal fragmentation induced by selenite exposure were greatly attenuated by this enzyme, supporting a chemical mediator role of superoxide for these processes. Despite a temporal relationship of AKT and ERK1/2 de-phosphorylation changes before the onset of PARP cleavage in MSeA-exposed cells, experiments with phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 did not show an enhancing effect of specific blocking of AKT on MSeA-induction of PARP cleavage. Taken together, exposure of DU145 cells to MSeA versus selenite induced differential patterns of cell cycle arrest and apoptosis execution as well as distinct patterns of effects on AKT, ERK1/2, JNK1/2, and p38MAPK phosphorylation and p27kip1 and p21cip1 expression. Multiple molecular pathways are likely differentially targeted by selenium metabolite pools to mediate cancer chemoprevention.