Rapid adaptation to central nervous system inhibitory effects of ethanol is observed in animals and humans and this acute functional tolerance (AFT) is influenced by genotype in rodents. Studies have been conducted to identify neurochemical processes influencing AFT to ethanol, but little is known regarding genetic regulation of AFT or genetic influences on processes that mediate acquisition of AFT. Our study was designed to develop, by selective breeding, lines of mice that differ in acquisition of AFT to ethanol; such mouse lines will be valuable in identifying the neuroadaptive processes mediating AFT. AFT is defined as the difference in blood ethanol concentration (BEC) at regaining balance on a stationary dowel rod after two consecutive doses of ethanol, 1.75 followed by 2.0 g/kg. Starting with a genetically heterogeneous foundation stock (HS/lbg), seven generations of selective breeding has been completed for high (HAFT1), low (LAFT1), and control lines and four generations have been completed for the replicate HAFT2 and LAFT2 lines. The lines do not differ in initial sensitivity to ethanol; however, the means for AFT scores differ by 2.3- and 4.3-fold for females and males, respectively (106.5 vs. 46.5 mg ethanol/dl blood) for females and 106.2 vs. 24.8 mg/dl for males). Frequency distributions for HAFT1 and LAFT1 show only modest overlap in AFT scores. The lines differ in rates of acquisition of AFT, but not in rates of ethanol clearance. Heritabilities were 0.04 and 0.26 for HAFT1 and LAFT1 lines, respectively, indicating that the selection was asymmetrical. Evidence is provided indicating that practice during intoxication has little effect on acquisition of AFT in HAFT1 and LAFT1 lines.