The muscarinic M₁ receptor (M₁R) is highly involved in cognition, and selective M₁ agonists have procognitive properties. Loss of M₁R has been found in postmortem brain tissue for several neuropsychiatric disorders and may be related to symptoms of cognitive dysfunction. ¹²³I-iododexetimide is used for imaging muscarinic acetylcholine receptors (mAchRs). Considering its high brain uptake and intense binding in M₁R-rich brain areas, ¹²³I-iododexetimide may be an attractive radiopharmaceutical to image M₁R. To date, the binding affinity and selectivity of ¹²³I-iododexetimide for the mAchR subtypes has not been characterized, nor has its brain distribution been studied intensively. Therefore, this study aimed to address these topics. The in vitro affinity and selectivity of ¹²⁷I-iododexetimide (cold-labeled iododexetimide), as well as its functional antagonist properties (guanosine 5′-[γ-³⁵S-thio]triphosphate [GTPγ³⁵S] assay), were assessed on recombinant human M₁R–M₅R. Distributions of ¹²⁷I-iododexetimide and ¹²³I-iododexetimide in the brain were evaluated using liquid chromatography–mass spectrometry and storage phosphor imaging, respectively, ex vivo in rats, wild-type mice, and M₁–M₅ knock-out (KO) mice. Inhibition of ¹²⁷I-iododexetimide and ¹²³I-iododexetimide binding in M₁R-rich brain areas by the M₁R/M₄R agonist xanomeline, or the antipsychotics olanzapine (M₁R antagonist) and haloperidol (low M₁R affinity), was assessed in rats ex vivo. In vitro, ¹²⁷I-iododexetimide displayed high affinity for M₁R (pM range), with modest selectivity over other mAchRs. In biodistribution studies on rats, ex vivo ¹²⁷I-iododexetimide binding was much higher in M₁R-rich brain areas, such as the cortex and striatum, than in cerebellum (devoid of M₁Rs). In M₁ KO mice, but not M₂–M₅ KO mice, ¹²⁷I-iododexetimide binding was strongly reduced in the frontal cortex compared with wild-type mice. Finally, acute administration of both an M₁R/M₄R agonist xanomeline and the M₁R antagonist olanzapine was able to inhibit ¹²³I-iododexetimide ex vivo, and ¹²³I-iododexetimide binding in M₁-rich brain areas in rats, whereas administration of haloperidol had no effect. The current results suggest that ¹²³I-iododexetimide preferentially binds to M₁R in vivo and can be displaced by M₁R ligands. ¹²³I-iododexetimide may therefore be a useful imaging tool as a way to further evaluate M₁R changes in neuropsychiatric disorders, as a potential stratifying biomarker, or as a clinical target engagement biomarker to assess M₁R.