Ticks introduce a variety of pharmacologically active molecules into their host during attachment and feeding in order to obtain a blood meal. People who are repeatedly exposed to ticks may develop an immune response to tick salivary proteins. Despite this response, people usually are unaware of having been bitten, especially if they are not repeatedly exposed to ticks. In order to develop a laboratory marker of tick exposure that would be useful in understanding the epidemiology of tick-borne infection and the immune response to tick bite, we developed an enzyme-linked immunosorbent assay (ELISA) to detect antibody to a recombinant form of calreticulin protein found in the salivary glands of Ixodes scapularis, a member of a complex of Ixodes ticks that serve as the vectors for Lyme disease, human babesiosis, and human granulocytic anaplasmosis. Using this assay, we tested sera obtained from C3H/HeN and BALB/c mice before and after experimental deer tick infestation. These mice developed antibody to Ixodes calreticulin antigen after infestation. We then used the same assay to test sera obtained from people before and after they experienced deer tick bite(s). People experiencing deer tick bite(s) developed Ixodes calreticulin-specific antibody responses that persisted for up to 17 months. This Ixodes recombinant calreticulin ELISA provides objective evidence of deer tick exposure in people.