Mutations in the “a” determinant of the surface gene have been associated with failure of hepatitis B immunoglobulin (HBIg) prophylaxis. We compared sequences from the surface and polymerase regions of hepatitis B virus (HBV) from 4 patients who failed high-dose HBIg therapy with two control groups: HBIg-treated patients who remained hepatitis B surface antigen (HBsAg)-negative (n = 4) and HBV-infected transplant recipients who never received HBIg (n = 4). Mutations within the surface and overlapping polymerase region were more common in patients failing HBIg than controls (P = .03), and mutations in the region of the “a” determinant were present only in patients failing HBIg. To examine the relationship between HBIg failure and duration of therapy, five additional treatment failures from a second transplantation center were sequenced (total with HBIg failure = 9). Mutations in the “a” determinant developed in 1 of 3 patients receiving HBIg for less than 6 months compared with 5 of 6 patients failing HBIg after 6 months of therapy (P = .23). The most frequently identified amino acid substitution was glycine to arginine at position 145 (present in 4 of 6 patients who failed HBIg after at least 6 months of treatment). A unique mutation within the YMDD motif (methionine to leucine) was present in 1 patient who failed HBIg treatment and who received a short course of ganciclovir. We conclude that the emergence of mutations in the “a” determinant accounts for some, but not all, treatment failures in patients receiving HBIg prophylaxis. Mutations in other regions of the S gene were more common in patients failing HBIg than controls, suggesting that domains other than the “a” determinant may be important.