The effects of the two central nervous system (CNS) depressant drugs ethanol and sodium valproate were compared using two pairs of mouse lines that had been selected from a heterogeneous stock for differential sensitivity to ethanol. The LS/SS lines differ in sensitivity to ethanol-induced sedation, and the WSP/WSR lines differ in the severity of their withdrawal convulsions after chronic ethanol treatment. We used these lines to test the hypothesis that ethanol and valproate act by the same mechanism. CNS depressant action was assessed by determining the brain drug concentration at which the mice lost their ability to balance on a stationary wooden dowel. LS mice were about twice as sensitive as SS mice to valproate-induced ataxia, in agreement with their reported relative sensitivity to ethanol. The WSR and WSP mice did not differ significantly in sensitivity to ethanol or valproate in this test. The intrinsic order and sensitivity to disordering of synaptosomal plasma membranes prepared from the four lines were measured using fluorescence polarization with the probe 1,6-diphenyl-1,3,5-hexatriene and EPR spectroscopy with 5-doxylstearic acid. No differences in the intrinsic membrane order of the four lines were detected with either technique. The sensitivities of the membranes from the four lines to ethanol- or valproate-induced disordering were not significantly different when measured by fluorescence polarization, but EPR spectroscopy revealed line differences in disordering sensitivity that correlated with the relative sensitivity of the four lines to the CNS depressant action of these drugs. These studies show that genetic factors modulate sensitivity to ethanol and valproate in a similar manner both in vivo and in vitro, suggesting that these drugs act by the same membrane-disordering mechanism.