The mouse is useful in studies of vascular biology because of its well-defined genetics and because the mouse genome can be manipulated. However, because only small amounts of mRNA can be extracted from blood vessels, the quantification of gene expression in individual mice is difficult. Endothelial NO synthase (eNOS) plays a major role in the regulation of vascular tone and growth. In addition, there appear to be sex differences in the production of NO under basal conditions in mouse aortas. The goals of this study were to develop a real-time polymerase chain reaction (PCR) method to quantify eNOS mRNA in blood vessels from mice and to examine eNOS mRNA levels in vessels from male and female mice. Blood vessels were isolated from C57BL/6 mice. Total RNA from individual mice was isolated and reverse-transcribed. The number of molecules of eNOS mRNA (after reverse transcription) was determined against cDNA standards, with 18S rRNA used as a control for RNA input and reverse-transcription efficiency. When expressed as copy numbers per nanogram of total RNA or as the ratio of eNOS to 18S rRNA, eNOS mRNA was lower in the aortas of female mice than in those of male mice at 7 to 9 months of age. In contrast, no difference in eNOS mRNA was found in the aortas of 2-month-old mice. In addition, eNOS mRNA levels were similar in the carotid, cerebral, and coronary arteries. These findings provide the first quantitative measurements of eNOS mRNA by using real-time PCR in the vessels of mice and suggest age- and sex-related differences in the basal levels of eNOS mRNA in mice. In addition, the eNOS region that was used for real-time PCR was amplified and sequenced for monkeys and other species. With modifications, this region may be used to design real-time PCR for eNOS in other species.