Lipid A, the biologically active component of lipopolysaccharide, stimulated nitric oxide (NO) production by isolated rat proximal tubules (as measured by NO2- release) in a time-dependent manner. At a concentration of 50 micrograms/ml, lipid A stimulated NO2- generation and guanosine 3',5'-cyclic phosphate (cGMP) production within 5 min. Both of these effects were blocked by NG-methyl-L-arginine (L-NMMA), an inhibitor of NO synthase or by 8-(N,N'-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular Ca++ release. Because an increase in NO production may be cytotoxic, we examined the cytotoxic potential of lipid A. At 90 min, lipid A (50 micrograms/ml) produced significant lactate dehydrogenase release (42 +/- 5%) compared to control (25 +/- 5%; P < .05). Both L-NMMA (1 mM) and TMB-8 (100 microM) completely protected against lipid A-induced cytotoxicity. TMB-8 but not L-NMMA inhibited the rise intracellular Ca++ concentration ([Ca++]i) in isolated proximal tubules elicited by lipid A. L-NMMA but not TMB-8 inhibited proximal tubule soluble NO synthase activity. Thus, in the proximal tubule, lipid A stimulates a rise in [Ca++]i that in turn activates constitutive NO synthase. Furthermore, these events lead ultimately to NO-dependent cytotoxicity. Therefore, these findings suggest the potential for lipopolysaccharide to have a direct impact on proximal tubule physiology and renal function in vivo and support the potential therapeutic benefits of NO synthase inhibitors in the treatment of endotoxemia.