Background and Purpose Galanin receptor subtype 1 (GALR1) and subtype 2 (GALR2) are G protein-coupled receptors (GPCRs) that mediate galanin's diverse physiological roles, including neurotransmission and neuronal modulation. Although both receptors share functional similarities, they exhibit distinct differences in signalling pathways. Whilst previous studies have focussed on galanin binding and G-protein selectivity, the role of plasma membrane-specific mechanisms, particularly cholesterol depletion's influence, remains unclear. This study investigates cholesterol's role in regulating trafficking of GALR1 and GALR2 and their function in live cells.Experimental Approach We employed real-time fluorescence techniques-Fluorescence Correlation Spectroscopy (FCS), Fluorescence Cross-Correlation Spectroscopy (FCCS), and Fluorescence Recovery After Photobleaching (FRAP)-to assess receptor-ligand interactions and lateral mobility in PC12 cells expressing EGFP-tagged GALR1 or GALR2.Key Results Both receptors were co-localised, co-trafficked and internalised with galanin, with receptor-peptide complexes dissociating prior to lysosomal degradation. Cholesterol selectively restricted GALR1's lateral diffusion and enhanced galanin binding and complex formation, whereas GALR2 remained unaffected. Interestingly, galanin binding relieved GALR1 from cholesterol-mediated restriction, increasing receptor mobility and suggesting a dynamic, cholesterol-dependent regulatory mechanism.Conclusions and Implications Cholesterol selectively modulates GALR1 trafficking and ligand interactions, whereas GALR2 operates independently of cholesterol, revealing distinct regulatory mechanisms for each receptor subtype. These findings provide new insights into the interplay between membrane composition and receptor function, with potential implications for developing targeted therapies for galanin-related disorders.