This experiment was designed to examine circadian rhythmicity of cortisol in saliva of growing pigs, in relation to age, gender, and (time of) stressor application. Additionally, the acute cortisol response to a stressor was studied. Five groups, each consisting of 3 barrows and 3 gilts, were involved in the experiment. In a Control Group, saliva samples were taken at 1-h intervals at 12, 16, 20, and 24 weeks of age. Within 1 week, rhythmicity of cortisol was assessed during two 24-h spans (Monday and Friday). Rhythm characteristics were evaluated by cosinor analysis, describing the rhythm by several parameters. In 2 groups at 12 weeks and 2 other groups at 20 weeks of age, a stressor was applied (4 h of isolation) on Thursday morning or evening. Again, rhythmicity was assessed on Monday and Friday by sampling at 2-h intervals. Acute cortisol effects were studied by sampling at several time-paints during isolation. Between 12 and 24 weeks of age, basal cortisol concentrations decreased and a rather stable and adult circadian rhythm was reached at 20 weeks of age. Average basal cortisol concentrations were higher in barrows than in gilts. Furthermore, after isolation, the amplitude of the rhythm was increased in barrows but was unchanged in gilts. The rhythm was more unstable and the maximum value tended to shift only after evening isolation. Stressor timing, but also age, was found to affect average cortisol concentrations. Moreover, stressor timing was important for the acute cortisol response: the increase was higher in the morning. The results of this study emphasize the importance of considering the circadian rhythmicity of cortisol, in relation to age, gender, and (time of) stressor application, when studying the cortisol response of animals to stressors.