Abstract Background Adenosine 5-triphosphate (ATP) and its breakdown products ADP and adenosine can act as extracellular messengers in a range of biological processes. Extracellular adenine nucleotides are metabolized by a number of enzymes including NTPDases and 5'-nucleotidase, which are considered to be the major regulators of purinergic signaling in the blood. Previous work by our group demonstrated that ATPase and ADPase activities in rat serum exhibit a 24-h temporal pattern, with higher enzyme activity during the dark (activity) phase. It was found that stress can cause disruptions in biological circadian rhythms and in the cardiovascular system. Therefore, the aim of the present study was to examine the influence of acute stress exposure upon temporal patterns of NTPDase and 5-nucleotidase enzyme activities in rat blood serum. Methods Adult male Wistar rats were divided into 4 groups: ZT0, ZT6, ZT12 and ZT18. Each group was subdivided in 4 groups: control, immediately, 6 h and 24 h after one hour of restraint stress. ATP, ADP and AMP hydrolysis were assayed in the serum. Results All stressed groups showed significant decreases in all enzyme activities at ZT 12 and ZT 18 when compared with control. Conclusion Acute stress provokes a decrease in nucleotidase activities dependent on the time that this stress occurs and this effect appears to persist for at least 24 hours. Stress can change levels of nucleotides, related to increased frequency of cardiovascular events during the activity phase. Altered levels of nucleotides in serum may be involved in cardiovascular events more frequent during the activity phase in mammals, and with their etiology linked to stress.