Gene-targeting technology is creating an explosion in the number of animals available with single gene mutations that affect the function of the central nervous system. Most gene-targeted mice are produced on a mixed genetic background of C57BL/6J and substrains of Strain 129. Understanding the behavioral characteristics and responses to various drugs of these parental strains is vital to interpreting data from gene-targeted mice. We directly compared C57BL/6J and Strain 129/SvJ mouse lines on several behavioral paradigms and in response to several hypnotic and anesthetic drugs. Compared to Strain 129/SvJ mice, C57BL/6J animals are more sensitive to the hypnotic effects of midazolam, zolpidem, and propofol, less sensitive to etomidate and ethanol, and do not differ in sensitivity to Ro15-4513 or pentobarbital. These strains do not differ in their sensitivity to the motor ataxic effects of the volatile anesthetics enflurane or halothane. However, Strain 129/SvJs are more sensitive to the immobilizing effects of halothane but not enflurane. Motor coordination differs initially, but with repeated testing strain differences are no longer apparent. Strain 129/SvJ mice are more anxious on the elevated plus maze and open-field activity assays. Thus, these mouse strains harbor polymorphisms that influence some, but not all, traits of interest to behavioral neuroscientists.