BACKGROUND: Early childhood multiple or persistent regulatory problems (RPs; crying, sleeping, or feeding problems) have been associated with a risk of behavioural problems in young adulthood. It has been suggested that this may be due to the possible influence of early RPs on the functioning of the hypothalamic-pituitary-adrenal (HPA) axis. However, associations between early RPs and HPA-axis activity in young adulthood remain unexplored. Thus, the aim of the current study was to investigate whether early childhood multiple or persistent RPs are associated with diurnal salivary cortisol in young adulthood. METHODS: At the ages of 5, 20 and 56 months, RPs of 308 children from the Arvo Ylppö Longitudinal Study were assessed via standardized parental interviews and neurological assessments. Multiple RPs were defined as two or three RPs at the age of 5 months and persistent RPs as at least one RP at 5, 20 and 56 months. At the mean age of 25.4 years (SD= 0.6), the participants donated saliva samples for cortisol at awakening, 15 and 30 min thereafter, 10:30 am, at noon, 5:30 pm, and at bedtime during one day. We used mixed model regressions, and generalized linear models for testing the associations, controlling for important covariates. RESULTS: Of the 308 children, 61 (19.8%) had multiple or persistent RPs in early childhood: 38 had multiple, and 27 had persistent RPs. Persistent RPs were associated with significantly higher cortisol peak and output in the waking period, and cortisol awakening response. On the other hand, multiple RPs were not associated with salivary cortisol. CONCLUSION: Children displaying persistent RPs throughout early childhood show, over two decades later, increased HPA axis activity in response to awakening stress. This may be one physiological mechanism linking early childhood RPs to adulthood behavioural outcomes.