Oxytocin and prolactin are potential candidates for the regulation of behavioral and physiological stress responses in the brain. To investigate the neurobiological basis of individual differences in stress responses in cattle, we examined the association of behavioral and hypothalamo-pituitary-adrenal axis reactivity to acute stressors and basal and stimulated levels of oxytocin and prolactin. Twenty Holstein heifer calves aged 2 weeks were subjected to a 10 min open-field test (OFT) followed by presentation of a feeding bucket for 15 min in the OF. If the calf contacted the bucket, a blast of air was applied to its muzzle (surprise test). Jugular blood samples collected before and after both tests were analyzed for oxytocin, prolactin, and cortisol. Relationships of basal and percent change in oxytocin or prolactin with behavioral responses in each test and percent change in cortisol were analyzed using principal components analysis and Spearman rank correlations. Plasma cortisol and prolactin concentrations were significantly elevated by the tests (p<0.005), but plasma oxytocin concentration did not significantly change (p>0.1). Four principal components explained 56.1% of the total variation: curiosity, general activity, fearfulness, and dependence on humans. Curiosity was inversely correlated with basal oxytocin level (rS=-0.683, p<0.05). General activity was positively correlated with prolactin reactivity (rS=0.448, p<0.05) and inversely with oxytocin reactivity to the novel environment (rS=-0.717, p<0.05). Fearfulness tended to correlate positively with basal oxytocin level (rS=0.583, p<0.1). Dependence on humans correlated with none of the hormonal parameters. The relationships of basal oxytocin level with curiosity and fearfulness for novel environments are of particular interest for future study.