Fourteen captive and five free-ranging Minnesota gray wolves (Canis lupus) were tested for the presence of rabies virus neutralizing antibodies (RVNA) after vaccination with an inactivated canine rabies vaccine. Blood was collected from all wolves prior to vaccination and at 1 mo postvaccination (PV) and from all captive and three wild wolves at 3 mo PV. In addition, one free-ranging wolf was sampled at 4 mo PV, and two free-ranging wolves were sampled at 6 mo PV. All wolves were seronegative prior to vaccination. RVNA were detected in 14 (100%) captive wolves and in four of five (80%) free-ranging wolves. The geometric mean titer of the captive wolves at 1 mo PV was significantly higher (P = 0.023) than in the free-ranging wolves. Five of 13 (38.5%) captive wolves and none of the three (0%) free-ranging wolves had measurable RVNA at 3 mo PV. No measurable RVNA were detected in the serum samples collected from the free-ranging wolves at 4 and 6 mo PV. These results should be interpreted with caution because of the small number of free-ranging wolves tested. Further research is needed to properly assess immune function and antibody response to vaccination in captive wolves in comparison with their free-ranging counterparts.