Abstract Locomotor activity is widely used to study nicotine effects, including genotypic differences, in rodents. In rats, chronic nicotine's (administered via osmotic minipump) effects on locomotion may differ based on animal strain, with Long–Evans rats more sensitive than Sprague–Dawley rats. Males and females also may differ in sensitivity. No studies, however, have compared males and females of the two strains. In addition, stress relief is a frequently cited reason for smoking, but the behavioral consequences of nicotine–stress interactions have rarely been examined. This experiment evaluated locomotor responses of male and female Sprague–Dawley and Long–Evans rats to 0, 6, or 12 mg/kg/day nicotine administered by minipump. Half of the animals in each drug condition were exposed to 20 min/day of immobilization stress to examine nicotine–stress interactions. Horizontal and vertical activities were measured on Drug Days 4 and 10. Stress effects were minimal and stress did not alter effects of nicotine. Nicotine (6 mg/kg/day) increased horizontal activity among Long–Evans but not among Sprague–Dawleys, with greater effects in Long–Evans females. Nicotine (6 mg/kg/day) increased vertical activity of all groups and 12 mg/kg/day decreased vertical activity of all groups except for Sprague–Dawley males. Results indicate that genotype and sex are relevant to understand nicotine's behavioral actions.