Salivary glands are vital to tick feeding success and also play a crucial role in tickbornepathogen transmission. In previous studies of Ixodes scapularis salivary glands,we demonstrated that saliva-producing type II and III acini are innervated byneuropeptidergic axons which release different classes of neuropeptides via theirterminals (Šimo et al., 2013, 2009b). Among these, the neuropeptide SIFamide—alongwith its cognate receptor—were postulated to control the basally located acinar valvevia basal epithelial and myoepithelial cells (Vancová et al., 2019). Here, we functionallycharacterized a second SIFamide receptor (SIFa_R2) from the I. scapularis genomeand proved that it senses a low nanomolar level of its corresponding ligand. InsectSIFamide paralogs, SMYamides, also activated the receptor but less effectivelycompared to SIFamide. Bioinformatic and molecular dynamic analyses suggestedthat I. scapularis SIFamide receptors are class A GPCRs where the peptide amidatedcarboxy-terminus is oriented within the receptor binding cavity. The receptor was foundto be expressed in Ixodes ricinus salivary glands, synganglia, midguts, trachea, andovaries, but not in Malpighian tubules. Investigation of the temporal expressionpatterns suggests that the receptor transcript is highly expressed in unfed I.ricinus female salivary glands and then decreases during feeding. In synganglia, asignificant transcript increase was detected in replete ticks. In salivary gland acini, anantibody targeting the second SIFamide receptor recognized basal epithelial cells,myoepithelial cells, and basal granular cells in close proximity to the SIFamidereleasingaxon terminals. Immunoreactivity was also detected in specific neuronsdistributed throughout various I. ricinus synganglion locations. The current findings,alongside previous reports from our group, indicate that the neuropeptide SIFamideacts via two different receptors that regulate distinct or common cell types in the basalregion of type II and III acini in I. ricinus salivary glands. The current study investigatesthe peptidergic regulation of the I. ricinus salivary gland in details, emphasizing thecomplexity of this system.