For several GPCRs, discrimination between agonism and antagonism is possible on the basis of thermodynamics parameters, such as binding enthalpy and entropy. In this study, we analyze whether agonists and antagonists can also be discriminated thermodynamically at the histamine H(1) receptor (H(1)R). Because previous studies revealed species differences in pharmacology between human H(1)R (hH(1)R) and guinea pig H(1)R (gpH(1)R), we analyzed a broad spectrum of H(1)R antagonists and agonists at hH(1)R and gpH(1)R. [(3)H]Mepyramine competition binding assay were performed at five different temperatures in a range from 283.15 to 303.15 K. In addition, we performed a temperature-dependent three-dimensional quantitative structure activity relationship study to predict binding enthalpy and entropy for histaprodifen derivatives, which can bind to H(1)R in two different orientations. Our studies revealed significant species differences in binding enthalpy and entropy between hH(1)R and gpH(1)R for some antagonists and agonists. Furthermore, in some cases, we found changes in heat capacity of the binding process that were different from zero. Differences in flexibility of the ligands may be responsible for this observation. For most ligands, the binding process to hH(1)R and gpH(1)R is clearly entropy-driven. In contrast, for the endogenous ligand histamine, the binding process is significantly enthalpy-driven at both species isoforms. Thus, a definite discrimination between antagonism and agonism based on thermodynamic parameters is possible for neither hH(1)R nor gpH(1)R, but thermodynamic analysis of ligand-binding may be a novel approach to dissect agonist- and antagonist-specific receptor conformations.