It is assumed that many G protein-coupled receptors (GPCRs) are restrained in an inactive state by the “ionic lock,” an interaction between an arginine in transmembrane domain (TM) 3 (R3.50) and a negatively charged residue in TM6 (D/E6.30). In the human histamine H₄ receptor (hH₄R), alanine is present in position 6.30. To elucidate whether this mutation causes the high constitutive activity of hH₄R, we aimed to reconstitute the ionic lock by constructing the A6.30E mutant. The role of R3.50 was investigated by generating hH₄R-R3.50A. Both mutants were expressed alone or together with Gα_i2 and Gβ₁γ₂ in Sf9 cells and characterized in GTPase, ³⁵S-labeled guanosine 5′-[γ-thio]triphosphate binding, and high-affinity agonist binding assays. Unexpectedly, compared with hH₄R, hH₄R-A6.30E showed only nonsignificant reduction of constitutive activity and G protein-coupling efficiency. The K_D of [³H]histamine was unaltered. By contrast, hH₄R-R3.50A did not stimulate G proteins. Thioperamide affinity at hH₄R-R3.50A was increased by 300 to 400%, whereas histamine affinity was reduced by approximately 50%. A model of the active hH₄R state in complex with the Gα_i2 C terminus was compared with the crystal structures of turkey β₁ and human β₂ adrenoceptors. We conclude that 1) constitutive activity of hH₄R is facilitated by the salt bridge D5.69-R6.31 rather than by the missing ionic lock, 2) Y3.60 may form alternative locks in active and inactive GPCR states, 3) R3.50 is crucial for hH₄R–G protein coupling, and 4) hH₄R-R3.50A represents an inactive state with increased inverse agonist and reduced agonist affinity. Thus, the ionic lock, although stabilizing the inactive rhodopsin state, is not generally important for all class A GPCRs.