Forty-seven Holstein calves (130 ± 3.43 kg of BW and 95 ± 1.5 d of age) were randomly assigned to 2 treatments [intact (INT), n = 23; or castrated (CAS), n = 24] to evaluate the effect of ring castration at 3 mo of age on welfare indicators. Castration was performed with local anesthesia (2% lidocaine, 3 mL in each testis and 2 mL in the scrotum) and analgesia (flunixin meglumine, intramuscularly, 3 mg/kg of BW). No local anesthesia or analgesia was used with INT calves. Serum cortisol concentration was determined at -120, 0, 30, 60, 90, and 180 min with respect to castration. At d 0, 1, 3, 7, 14, 21, 28, 35, 42, and 49, serum haptoglobin concentration was determined, rectal body and scrotal temperatures were measured, lesions at the castration site were scored, and the activity and behavior of 18 calves (9 INT and 9 CAS) were recorded continuously for 24 h. Weekly BW and concentrate and straw DMI were recorded. To evaluate humoral immunity, at 14 d after castration, ovalbumin was injected subcutaneously and serum antibody titers against ovalbumin before the injection and at d 35 were determined. At d 49 after castration, calves were intravenously injected with ACTH, and at 0, 1, 2, and 4 h thereafter, serum cortisol and testosterone concentrations were determined. Average daily gain was greater (P < 0.001) in INT than CAS calves (1.36 vs. 1.16 ± 0.038 kg/d, respectively). Area under the curve of cortisol at castration day was reduced (P < 0.05) in CAS calves compared with INT calves (18 vs. 33 ± 5.2 nmol/L per hour, respectively). The main scrotal lesion score observed in CAS calves throughout the study was 0, corresponding to no visible swelling, inflammation, or infection. However, scrotal lesion scores classified as 1 (swelling) were greater (P < 0.01) at 21 and 28 d after castration than at 1, 3, 7, and 14 d. Abnormal standing occurred more frequently (P < 0.001) in CAS than INT calves (2.6 vs. 0.5 ± 0.03%, respectively) from 3 to 14 d after castration. Head turning tended (P = 0.06) to be greater at d 14 of the study in CAS than INT calves (3.0 vs. 2.6 ± 0.04%, respectively). At d 49, 100% of CAS calves had no testes and no serum testosterone was detected. In summary, ring castration of Holstein calves performed at 3 mo of age with local anesthesia and analgesia decreased ADG and affected some behavioral traits during the first 14 d after castration. However, intake, serum cortisol and haptoglobin concentrations, rectal temperature, and humoral immunity were not altered. ©2010 American Society of Animal Science.