Nitric Oxide (NO) can be cytotoxic or cytoprotective depending on amount and location of its generation. eNOS is important in modulating blood flow and is allosterically regulated. Inducible NOS (iNOS) tends to produce large quantities of NO leading to cell injury. We studied the role and regulation of NOS in carbon tetrachloride (CCl4)-induced hepatotoxicity in newborn rats. eNOS was expressed before birth, significantly increased on day of life (DOL) 2 reaching a maximum at DOL-20. iNOS was absent at all ages. CCl4 treatment resulted in hepatic injury in newborn rats and damage was intensified by co-administration of a general NOS inhibitor. CCl4 treatment increased eNOS activity without change in mRNA or protein levels. Administration of CCl4 resulted in an increase in phosphorylation of threonine protein kinase (Akt) and eNOS, associated with an increase in eNOS activity. Administration of wortmannin (phosphatidylinositol 3-kinase, PI3 K, inhibitor) attenuated the phosphorylation of Akt and eNOS and reduced eNOS activity. Co-administration of CCl4 and wortmannin potentiated the degree of hepatic injury. iNOS was not detectable in CCl4-treated rats. This data indicates a protective role for eNOS in CCl4-induced hepatotoxicity in newborn rats with protection accomplished by activation of eNOS via posttranslational modification of the PI3 K/Akt signaling pathway.