Development of specific IgE antibodies to the oligosaccharide galactose-α-1, 3galactose (α-gal) following tick bites has been shown to be the source of red meat allergy. In this study, we investigated the presence of α -gal in four tick species: the lonestar tick (Amblyomma americanum), the Gulf-Coast tick (Amblyomma maculatum), the American dog tick (Dermacentor variabilis), and the black-legged tick (Ixodes scapularis) by using a combination of immunoproteome, carbohydrate analysis, and basophil activation approaches. Using anti-α-gal antibodies, α -gal was identified in the salivary glands of both Am. americanum and Ix. scapularis, while Am. maculatum and De. variabilis appeared to lack the sugar. PNGase F treatment confirmed the deglycosylation of N-linked α -gal-containing proteins in tick salivary glands. Immunolocalization of α -gal moieties to the salivary secretory vesicles of the salivary acini also confirmed the secretory nature of α -gal-containing antigens in ticks. Am. americanum ticks were fed human blood (lacks α -gal) using an artificial membrane feeding system to determine the source of α -gal. N-linked glycan analysis revealed that Am. americanum and Ix. scapularis have α -gal in their saliva and salivary glands, but Am. maculatum contains no detectable quantity. Salivary samples from Am. americanum and Ix. scapularis stimulated activation of basophils primed with plasma from α -gal allergic subjects. Together, our data support the idea that bites from certain tick species may specifically create a risk for the development of α -gal-specific IgE and hypersensitivity reactions in humans. Alpha-Gal syndrome challenges the current food allergy paradigm and broadens opportunities for future research.