Abstract Purpose Pridopidine is an investigational drug for Huntington disease (HD). Pridopidine was originally thought to act as a dopamine stabilizer. However, pridopidine shows highest affinity to the sigma-1 receptor (S1R) and enhances neuroprotection via the S1R in preclinical studies. Using [¹⁸F] fluspidine and [¹⁸F] fallypride PET, the purpose of this study was to assess in vivo target engagement/receptor occupancy of pridopidine to the S1R and dopamine D2/D3 receptor (D2/D3R) at clinical relevant doses in healthy volunteers (HVs) and as proof-of-concept in a small number of patients with HD. Methods Using [¹⁸F] fluspidine PET (300 MBq, 0–90 min), 11 male HVs (pridopidine 0.5 to 90 mg; six dose groups) and three male patients with HD (pridopidine 90 mg) were investigated twice, without and 2 h after single dose of pridopidine. Using [¹⁸F] fallypride PET (200 MBq, 0–210 min), four male HVs were studied without and 2 h following pridopidine administration (90 mg). Receptor occupancy was analyzed by the Lassen plot. Results S1R occupancy as function of pridopidine dose (or plasma concentration) in HVs could be described by a three-parameter Hill equation with a Hill coefficient larger than one. A high degree of S1R occupancy (87% to 91%) was found throughout the brain at pridopidine doses ranging from 22.5 to 90 mg. S1R occupancy was 43% at 1 mg pridopidine. In contrast, at 90 mg pridopidine, the D2/D3R occupancy was only minimal (~ 3%). Conclusions Our PET findings indicate that at clinically relevant single dose of 90 mg, pridopidine acts as a selective S1R ligand showing near to complete S1R occupancy with negligible occupancy of the D2/D3R. The dose S1R occupancy relationship suggests cooperative binding of pridopidine to the S1R. Our findings provide significant clarification about pridopidine’s mechanism of action and support further use of the 45-mg twice-daily dose to achieve full and selective targeting of the S1R in future clinical trials of neurodegenerative disorders. Clinical Trials.gov Identifier: NCT03019289 January 12, 2017; EUDRA-CT-Nr. 2016-001757-41.