1. Drinking behaviour after water deprivation is one of the standard tests used to study thirst in humans and animals. Diurnal cycle and food availability are known to influence water intake, but have not been considered in previous studies of thirst after water deprivation. In the present study, we examined the effects of diurnal variation and food availability on water intake after 24 h water deprivation in rats. 2. All rats cycled through four treatments in varying order. These treatments were: (i) 24 h water deprivation with free access to food from 1900 h one day to 1900 h the next day, followed by free access to both food and water (Night-with-Food); (ii) 24 h water deprivation with free access to food from from 1900 h one day to 1900 h the next day, followed by free access to water but not food (Night-without-Food); (iii) 24 h water deprivation with free access to food from 0700 h one day to 0700 h the next day, followed by free access to both food and water (Day-with-Food); or (iv) 24 h water deprivation with free access to food from 0700 h one day to 0700 h the next day, followed by free access to water but not food (Day-without-Food). The amount of water consumed during the first 6 h, post-24 h water deprivation, was examined under each condition. 3. There was a significant diurnal effect (P < 0.001) and a significant food availability effect (P = 0.007) on the water consumed in the 6 h period after water deprivation. Most water was consumed by the Night-with-Food group and the least amount of water was consumed by the Day-without-Food group. These effects persisted after correction for water intake during 6 h periods from 0700 and 1900 h with and without food but without previous water deprivation. The diurnal and food availability effects on water consumption were independent (P = 0.5). 4. The coefficient of variability for each group suggests that the most sensitive measurements of water intake are obtained during the day in the absence of food. 5. We conclude that both the time of day and access to food independently alter water intake in rats subjected to a previous 24 h water deprivation. Our study also supports the validity of performing water intake measurements in thirst studies in rats during the day.