We assessed the effect of timing and other biological variables on immune response among health care workers (HCW) vaccinated with hepatitis B vaccine. A total of 2.058 HCW received three doses and were tested for anti-HBs within 6 months. 92.2% of the HCW had evidence of seroprotection. Multivariable analysis showed that controlling for age, the estimated non-response OR associated with a delayed second dose was 2.16 (95% CI: 1.46, 3.18, p=0.004). We found a decreasing response rate with increasing age. Particular attention should be given to those HCW who are late for the second vaccine dose and to older subjects.