Keap1 is a key regulator of the Nrf2 transcription factor, which transactivates the antioxidant response element (ARE) and upregulates numerous proteins involved in antioxidant defense. Under basal conditions, Keap1 targets Nrf2 for ubiquitination and proteolytic degradation and as such is responsible for the rapid turnover of Nrf2. In response to oxidants and electrophiles, Nrf2 is stabilized and accumulates in the nucleus. The mechanism for this effect has been proposed to involve thiol-dependent modulation of Keap1 leading to loss of its ability to negatively regulate Nrf2. We have previously shown that nitric oxide and S-nitrosothiols cause nuclear accumulation of Nrf2 and upregulation of the ARE-regulated gene HO-1. Here we show that nitric oxide and S-nitrosocysteine (CSNO) cause time- and dose-dependent Keap1 thiol modification. These studies were carried out in HEK293 cells and in HEK293 cells overexpressing hemagglutinin-tagged Keap1. Furthermore we demonstrate that in response to CSNO Keap1 accumulates in the nucleus with a time course similar to that of Nrf2.