The importance of the dynamic interplay between the opioid and the serotonin neuromodulatory systems in chronic pain is well recognized. In this study, we investigated whether these two signalling pathways can be integrated at the single-cell level via direct interactions between the mu-opioid (MOP) and the serotonin 1A (5-HT) receptors. Using fluorescence cross-correlation spectroscopy (FCCS), a quantitative method with single-molecule sensitivity, we characterized in live cells MOP and 5-HT interactions and the effects of prolonged (18 h) exposure to selected non-peptide opioids: morphine, codeine, oxycodone and fentanyl, on the extent of these interactions. The results indicate that in the plasma membrane, MOP and 5-HT receptors form heterodimers that are characterized with an apparent dissociation constant Kdapp = (440 ± 70) nM). Prolonged exposure to all non-peptide opioids tested facilitated MOP and 5-HT heterodimerization and stabilized the heterodimer complexes, albeit to a different extent: Kd,&nbsp;Fentanylapp = (80 ± 70) nM), Kd,Morphineapp = (200 ± 70) nM, Kd,&nbsp;Codeineapp = (100 ± 70) nM and Kd,&nbsp;Oxycodoneapp = (200 ± 70) nM. The non-peptide opioids differed also in the extent to which they affected the mitogen-activated protein kinases (MAPKs) p38 and the extracellular signal-regulated kinase (Erk1/2), with morphine, codeine and fentanyl activating both pathways, whereas oxycodone activated p38 but not ERK1/2. Acute stimulation with different non-peptide opioids differently affected the intracellular Ca levels and signalling dynamics. Hypothetically, targeting MOP-5-HT heterodimer formation could become a new strategy to counteract opioid induced hyperalgesia and help to preserve the analgesic effects of opioids in chronic pain.