BACKGROUND The serotonergic and opioidergic neurotransmitter systems are critical regulators of the hypothalamic-pituitary-adrenal (HPA) axis through their respective excitatory and inhibitory inputs. Serotonin transporter (5-HTT) genotype has been studied as a marker of HPA axis dysregulation and for predicting risk of psychopathology, with mixed findings. OBJECTIVE We stimulated the HPA axis with naloxone, an opioid receptor antagonist, to examine cortisol reactivity based on 5-HTT-linked polymorphic region (5-HTTLPR) genotypes. METHODS Healthy community volunteers (N = 78) received intravenous (IV) placebo followed by sequential doses of IV naloxone (50, 100, 200, and 400 μg/kg) every 30 min. Plasma cortisol was measured every 15 min. Participants were genotyped for the long (L) and short (S) alleles of the 5-HTT gene and for rs25531 (A/G) in the 5-HTTLPR repetitive element and compared by the 5-HTTLPR/rs25531 genotype (triallele) and by the 5-HTTLPR genotype (biallele) classification. RESULTS In triallele analyses, individuals with one or more L(A) alleles showed higher cortisol response to naloxone compared with individuals with no L(A) alleles. In biallele analyses, less robust effects were found, although individuals with two L alleles showed a higher cortisol response compared with other genotypes. CONCLUSIONS Naloxone blockade leads to a greater activation of the HPA axis among individuals with the L(A) allele. Including rs25531 in the analysis with the 5-HTTLPR genotype appears more sensitive in detecting genetic differences in naloxone-induced cortisol than when using only the 5-HTTLPR genotype. Future research should investigate the interactive effects between the serotonergic and opioidergic systems on HPA axis dysregulation and psychopathophysiology.