Background Serotonergic brain signaling is considered critical for an appropriate and dynamic adaptation to stress, seemingly through modulating limbic system functions, such as the hypothalamicpituitary- adrenal (HPA)-axis. This interplay is of great interest since it holds promise as a target for preventing stress-related brain disorders, e.g., major depression. Our group has previously observed that prefrontal serotonin transporter (5-HTT) binding, imaged with positron emission tomography (PET), is positively associated with the cortisol awakening response (CAR), an index of HPA axis stress hormone dynamics. The aim of this cross-sectional study was to replicate the previous finding in a larger independent group of healthy individuals. Methods Molecular imaging and cortisol data were available for 90 healthy individuals. Prefrontal 5- HTT binding was imaged with [11C]DASB brain PET. Non-displaceable 5-HTT binding potential (BPND) was quantified using the Multilinear Reference Tissue Model 2 (MRTM2) with cerebellum as the reference region. CAR was based on five serial salivary cortisol samples within the first hour upon awakening. The association between CAR and prefrontal 5- HTT BPND was evaluated using a multiple linear regression model adjusted for age and sex. Further, we tested for sex differences in the association. Finally, an exploratory analysis of the association, was performed in 8 additional brain regions. Results We observed no statistically significant association between 5-HTT binding and CAR corrected for age and sex in the prefrontal cortex (β = -0.28, p = 0.26). We saw no interaction with sex on the association (p = 0.99). Conclusion We could not confirm a positive association between CAR and prefrontal 5-HTT BPND in this independent dataset. Also, sex differences in the association were not apparent. Our data do not exclude that the serotonin transporter system is involved in the regulation of stress responses in at-risk or manifest depressed states.