The aim of this study was to test the effects of mother rearing on behavioural and physiological stress reactions of calves in challenging situations. Thus, we compared mother-reared and artificially reared calves that were kept in the same group but with varying contact with adults. Mother-reared calves (Mother) were suckled and had unrestricted contact with their mothers and also with the cow herd in the cubicle barn; artificially reared calves were fed milk up to 16 kg per day and animal via an automatic milk feeder (Automat). At the age of 43 days, the calves were separated from the group for 15 min (isolation test; Mother: n = 16; Automat: n = 16), and at 90 days of age, they underwent a social confrontation test with an unfamiliar calf in an arena for 20 min (Mother: n = 11; Automat: n = 11). Data were analysed using ANOVA and GLMM. In the isolation test, Mother calves showed more (P < 0.05) escape behaviour and tended to be more vigilant (P < 0.1). Concerning physiological parameters, no differences were detected in the mean heart rate over 15 min of isolation, but the increase in salivary cortisol concentrations 5 min after the end of the test tended to be lower in Mother calves than in Automat calves (P < 0.1). During confrontation, Mother calves showed less frequently solitary play behaviour (i.e. mainly locomotor play) than Automat calves (P < 0.05) but initiated more frequently social play when no cow was present adjacent to the test arena (P < 0.05). The results suggest that mother-reared calves showed higher motivation to rejoin their mothers and/or herd and tried to cope more actively with being isolated. In addition, in the confrontation test Mother calves seemed to be socially more active and more attentive to their social environment, but less motivated for locomotor play possibly due to the much larger space available to them in the cow barn.