MK6240 is a second-generation tau PET tracer designed to detect the neurofibrillary tangles in the brains of patients with Alzheimer's disease (AD). The aim of the study was to characterize 3H-MK6240 in AD and control brain tissue and to compare its binding properties with those of first-generation tau PET tracers. Saturation binding assays with 3H-MK6240 were carried out in the temporal and parietal cortices of AD brains to determine the maximum number of binding sites (Bmax) and the dissociation constants (Kd) at these sites. Competitive binding assays were carried out between 3H-MK6240 and unlabelled MK6240, AV-1451 (aka T807, flortaucipir) and THK5117, and between 3H-THK5351 and unlabelled MK6240. Regional binding studies with 3H-MK6240 were carried out in homogenates from six AD and seven control brains and, using autoradiography, on large frozen sections from two AD brains and one control brain. The saturation binding assays gave Bmax and Kd values of 59.2 fmol/mg and 0.32 nM in the temporal cortex and 154.7 fmol/mg and 0.15 nM in the parietal cortex. The competitive binding assays revealed two binding sites with affinities in the picomolar and nanomolar range shared by 3H-MK6240 and all the tested unlabelled compounds. There were no binding sites in common between 3H-THK5351 and unlabelled MK6240. Regional binding of 3H-MK6240 was significantly higher in AD brain tissue than in controls. Binding in brain tissue from AD patients with early-onset AD was significantly higher than in brain tissue from patients with late-onset AD. Binding of 3H-MK6240 was not observed in off-target regions. Autoradiography showed high regional cortical binding in the two AD brains and very low binding in the control brain. 3H-MK6240 has a high binding affinity for tau deposits in AD brain tissue but also has different binding characteristics from those of the first-generation tau tracers. This confirms the complexity of tau tracer binding on tau deposits with different binding affinities for different binding sites.