Sex differences in exploratory and spontaneous behaviour were investigated in 65 male and 67 female control CD1 mice of F1-generation in two-generation toxicity studies over a 6-year-period. Exploratory and spontaneous behaviour was measured using an animal movement analysing system (SCANET CV-40; Melquest Ltd.) consisted of two crossing sensor frames of 72 units of detectors of near-infrared photosensors for measuring motor activity. Exploratory behaviour of F1-generation mice was investigated at 8 weeks of age. Adult females showed less activity in the rearing time and average rearing time than adult males in the 10-minute total. These variables indicated similar longitudinal patterns in male and female mice during the 10-minute observation period and indicated the significant distance between the male and female lines. Spontaneous behaviour of F1-generation mice was investigated at 9–10 weeks of age. No significant differences showed between male and female mice in all measured variables in the 120-minute total. The average rearing time indicated a similar longitudinal pattern between male and female mice during the 120-minute observation period. Other variables indicated a different longitudinal pattern between male and female mice. In the relationship between exploratory and spontaneous behaviour, most variables of males excluding the number of horizontal activities showed significant but weak correlations. In females, most variables excluding the number of horizontal activities and movement time showed significant but weak correlations between exploratory and spontaneous behaviour. In males, the activity levels of exploratory behaviour correlated with spontaneous behaviour levels, but females indicated lower correlations than males. From these results, it appears that female CD1 mice display shorter rearing time of exploratory behaviour than male CD1 mice, and the longitudinal patterns of spontaneous behaviour indicate sex differences.