Our objective was to determine if repeated exposure of lactating dairy cows to human chorionic gonadotropin (hCG) would induce an antibody (Ab) response against hCG. Cows either received an hCG injection (hCG; n = 24, each given 2000 IU im) or no treatment (CON; n = 22) 18 days after a timed AI (TAI) and 7 days before initiation of Ovsynch for resynchronization of ovulation and TAI. A subgroup of cows continued in the experiment to receive a second hCG injection (n = 17) 35 days after the first exposure to hCG, whereas another subgroup served as controls (n = 9). Another subgroup of cows continued in the experiment to receive a third hCG injection (n = 11) 35 days after the second exposure to hCG, whereas cows not receiving hCG served as controls (n = 8). A binding radioimmunoassay was used to detect hCG antibodies in serum samples collected 0, 7, 14, 21, and 28 days after treatment. A positive Ab response (>6.2% bound) was defined as three standard deviations above CON binding. No cows had hCG antibodies at Day 0 before the first exposure to hCG. After the first hCG treatment, there was no difference (P = 0.52) between Ab positive cows in CON (0%) and hCG (4%) treatments. At the second hCG treatment, on Day 0 there was no difference (P = 0.65) between CON (0%) and hCG (6%) cows, whereas, more (P = 0.02) hCG cows (47%) were positive than CON cows (0%) within 28 days of the hCG injection. At the third hCG injection, hCG cows tended (P = 0.09) to have a greater percentage of Ab positive (36%) than CON cows (0%), whereas after the injection, a greater (P < 0.01) percentage of hCG cows were positive (hCG = 73% vs. CON = 0%). After the second and third exposure to hCG, 8 of 17 and 8 of 11 cows within the hCG group had greater percent Ab bound at 7, 14, 21, and 28 days after hCG than cows in CON and those with no Ab response. The greatest percent Ab binding occurred at 14 days after the second and third hCG exposure. We concluded that some but not all lactating dairy cows developed an Ab response after repeated exposure to hCG and that maximum response occurred within 14 days after hCG exposure.