Nitric oxide (NO) is suggested to play a role in liver injury elicited by acetaminophen (APAP). Hepatic microcirculatory dysfunction also is reported to contribute to the development of the injury. As a result, the role of NO in hepatic microcirculatory alterations in response to APAP was examined in mice by in vivo microscopy. A selective inducible nitric oxide synthase (iNOS) inhibitor, L-N⁶-(1-iminoethyl)-lysine (L-NIL), or a non-selective NOS inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME), were intraperitoneally administered to animals 10 min before APAP gavage. L-NIL suppressed raised alanine aminotransferase (ALT) values 6 hours after APAP, whereas L-NAME increased those 1.7-fold. Increased ALT levels were associated with hepatic expression of iNOS. L-NIL, but not L-NAME reduced the expression. APAP caused a reduction (20%) in the numbers of perfused sinusoids. L-NIL restored the sinusoidal perfusion, but L-NAME was ineffective. APAP increased the area occupied by infiltrated erythrocytes into the extra-sinusoidal space. L-NIL tended to minimize this infiltration, whereas L-NAME further enhanced it. APAP caused an increase (1.5-fold) in Kupffer cell phagocytic activity. This activity in response to APAP was blunted by L-NIL, while L-NAME further elevated it. L-NIL suppressed APAP-induced decreases in hepatic gluthatione levels. These results suggest that NO derived from the inducible isoform of NOS contributes to APAP-induced parenchymal cell injury and hepatic microcirculatory disturbances. L-NIL exerts preventive effects on the liver injury partly by inhibiting APAP bioactivation. In contrast, NO derived from constitutive isoforms of NOS exerts a protective role in liver microcirculation against APAP intoxication, and thereby minimizes liver injury.