A young child's stress-sensitive neurobiological system is immature and open to being shaped by experience. When children enter preschool, external demands on them to adjust are different from the demands they experience at day care. In Finland, the last year before children transfer to the comprehensive school is called preschool. The preschool year is far more academically demanding than earlier day care years, emphasizing school-like activities along with age-appropriate social and cognitive challenges. The first aim of the present study was to study whether the preschool year (six-year-old children) has an effect on the children's cortisol reactivity. To examine the changes in stress regulation, cortisol reactivity was investigated both in the autumn and in the spring. The second aim of the study was to explore whether demographic factors, temperament and the quality of preschool environment have an effect on the daily variation of the cortisol level among the participants of the study. Five day care centres in metropolitan Helsinki, Finland, participated in this study and 91 six-year-old preschool children served as participants. Our results showed that the children displayed typical cortisol reactivity during the autumn as expected, whereas their cortisol values were significantly higher in the spring. The autumn measurements revealed a significant relationship between the cortisol effects and the temperament characteristics of sadness, anger/frustration and perceptual sensitivity. These characteristics were not as evident when measured during the spring. Additionally, the results indicated that quality of learning environment affects changes in children's cortisol reactivity.