The histamine H3 receptor is a G protein-coupled receptor (GPCR) drug target that is highly expressed in the CNS, where it acts as both an auto- and hetero-receptor to regulate neurotransmission. As such, it has been considered as a relevant target in disorders as varied as Alzheimer's disease, schizophrenia, neuropathic pain and attention deficit hyperactivity disorder. A range of competitive antagonists/inverse agonists have progressed into clinical development, with pitolisant approved for the treatment of narcolepsy. Given the breadth of compounds developed and potential therapeutic indications, we assessed the comparative pharmacology of six investigational histamine H3 agents, including pitolisant, using native tissue and recombinant cells. Whilst all of the compounds tested displayed robust histamine H3 receptor inverse agonism and did not differentiate between the main H3 receptor splice variants, they displayed a wide range of affinities and kinetic properties, and included rapidly dissociating (pitolisant, S 38093-2, ABT-239) and slowly dissociating (GSK189254, JNJ-5207852, PF-3654746) agents. S 38093-2 had the lowest histamine H3 receptor affinity (pKB values 5.7–6.2), seemingly at odds with previously reported, potent in vivo activity in models of cognition. We show here that at pro-cognitive and anti-hyperalgesic/anti-allodynic doses, S 38093-2 preferentially occupies the mouse sigma-1 receptor in vivo, only engaging the histamine H3 receptor at doses associated with wakefulness promotion and neurotransmitter (histamine, ACh) release. Furthermore, pitolisant, ABT-239 and PF-3654746 also displayed appreciable sigma-1 receptor affinity, suggesting that this property differentiates clinically evaluated histamine H3 receptor antagonists and may play a role in their efficacy.