We have extended our previous findings and shown that human immunodeficiency virus Tat protein, in addition to nitric oxide (NO), stimulated rat microglial cultures to release pro-inflammatory cytokine interleukin-1beta and tumour necrosis factor-alpha in a nuclear factor (NF)-kappaB-dependent manner. At the same time, Tat stimulated the accumulation of free radicals, as indicated by the increased levels of isoprostane 8-epi-prostaglandin F(2alpha) (8-epi-PGF(2alpha)), a reliable marker of lipid peroxidation and oxidative stress, by a mechanism unrelated to NF-kappaB activation. The presence of free radical scavengers abrogated Tat-induced 8-epi-PGF(2alpha) accumulation without affecting NO and cytokine production. Consistently, Tat-induced IkappaBalpha degradation - an index of NF-kappaB activation - was not affected by free radical scavengers, but was prevented by an NF-kappaB-specific inhibitor. Our observations indicate that NF-kappaB plays a key role in Tat-dependent microglial activation, and that oxidative stress and NF-kappaB activation induced by Tat occur by independent mechanisms.