The α7 nicotinic acetylcholine receptor (α7 nAChR) is involved in several cognitive and physiologic processes; its expression levels and patterns change in neurologic and psychiatric diseases, such as schizophrenia and Alzheimer’s disease, which makes it a relevant drug target. Development of selective radioligands is important for defining binding properties and occupancy of novel molecules targeting the receptor. We tested the in vitro binding properties of [¹²⁵I]Iodo-ASEM [(3-(1,4-diazabycyclo[3.2.2]nonan-4-yl)-6-(¹²⁵I-iododibenzo[b,d]thiopentene 5,5-dioxide)] in the mouse, rat and pig brain using autoradiography. The in vivo binding properties of [¹⁸F]ASEM were investigated using positron emission tomography (PET) in the pig brain. [¹²⁵I]Iodo-ASEM showed specific and displaceable high affinity (~1 nM) binding in mouse, rat, and pig brain. Binding pattern overlapped with [¹²⁵I]α-bungarotoxin, specific binding was absent in α7 nAChR gene-deficient mice and binding was blocked by a range of α7 nAChR orthosteric modulators in an affinity-dependent order in the pig brain. Interestingly, relative to the wild-type, binding in β2 nAChR gene-deficient mice was lower for [¹²⁵I]Iodo-ASEM (58% ± 2.7%) than [¹²⁵I]α-bungarotoxin (23% ± 0.2%), potentially indicating different binding properties to heteromeric α7β2 nAChR. [¹⁸F]ASEM PET in the pig showed high brain uptake and reversible tracer kinetics with a similar spatial distribution as previously reported for α7 nAChR. Blocking with SSR-180,711 resulted in a significant decrease in [¹⁸F]ASEM binding. Our findings indicate that [¹²⁵I]Iodo-ASEM allows sensitive and selective imaging of α7 nAChR in vitro, with better signal-to-noise ratio than previous tracers. Preliminary data of [¹⁸F]ASEM in the pig brain demonstrated principal suitable kinetic properties for in vivo quantification of α7 nAChR, comparable to previously published data.