Species isoforms of histamine H2-, H3-, and H4-receptors differ in their pharmacological properties. The study aim was to dissect differences between the human H1R (hH1R) and guinea pig H1R (ghH1R). We coexpressed hH1R and gpH1R with regulators of G-protein signaling in Sf9 insect cells and analyzed the GTPase activity of Gq-proteins. Small H1R agonists showed similar effects at hH1R and gpH1R, whereas bulkier 2-phenylhistamines and histaprodifens were up to approximately 10-fold more potent at gpH1R than at hH1R. Most 2-phenylhistamines and histaprodifens were more efficacious at gpH1R than at hH1R. Several first-generation H1R antagonists were approximately 2-fold, and arpromidine-type H1R antagonists up to approximately 10-fold more potent at gpH1R than at hH1R. [3H]Mepyramine competition binding studies confirmed the potency differences of the GTPase studies. Phe-153-->Leu-153 or Ile-433-->Val-433 exchange in hH1R (hH1R-->gpH1R) resulted in poor receptor expression, low [3H]mepyramine affinity, and functional inactivity. The Phe-153-->Leu-153/Ile-433-->Val-433 double mutant expressed excellently but only partially changed the pharmacological properties of hH1R. Small H1R agonists and 2-phenylhistamines interacted differentially with human and guinea pig H2R in terms of potency and efficacy, respectively. Our data show the following: 1) there are differences in agonist- and antagonist-pharmacology of hH1R and gpH1R encompassing diverse classes of bulky ligands. These differences may be explained by higher conformational flexibility of gpH1R relative to hH1R; 2) Phe-153 and Ile-433 are critical for proper folding and expression of hH1R; and 3) H2R species isoforms distinguish between H1R agonists.