It is thought that the salivary cortisol awakening response can serve as a reliable marker of hypothalamic-pituitary-adrenocortical activity. Response magnitude might be influenced by stress, but results of studies on work-related stress have been inconclusive. Non-compliance with the sampling schedule is a potential confounding factor that has rarely been controlled. The objective of the study was to determine whether the cortisol awakening response is greater on a work day than on a weekend day, and whether responses vary with gender and socioeconomic position. Compliance was controlled by excluding participants who reported a delay of more than 10 min between waking up and taking the first saliva sample. Data were collected from 196 men and women aged 47 to 59 years drawn from the Whitehall II cohort of British civil servants, with free salivary cortisol sampled immediately after waking up and 30 min later on 2 days. Data on stress, perceived control and happiness over the remainder of the day, and on sleep quality, time of waking, and health behaviour were also obtained. The awakening response was lower in non-compliant participants than in compliant ones, so non-compliant participants were excluded from further analyses. Salivary cortisol levels on waking did not differ by gender or socioeconomic position, or between work and weekend days. However, the cortisol awakening response (defined as the difference between waking and 30 min later) was greater on work than weekend days (mean increases 10.5 and 3.7 nmol/l, P < 0.001). On the work day, women showed larger increases than men (P = 0.011), but there were no gender differences on the weekend day. Across both days, lower socioeconomic position was associated with a larger cortisol awakening response (P = 0.014). Time of waking up was not related to the cortisol awakening response on either day. Participants rated themselves as more stressed, less in control, and less happy over the remainder of the work than weekend day. These results indicate that the cortisol awakening response occurs on both work and non-work days, but that anticipation of the working day is associated with an enhanced response. Cortisol output over the early part of the day may be particularly sensitive to the influence of chronic stress and its anticipation, especially in women.