An antigen-specific lymphoblastogenesis assay for duck hepatitis B surface antigen (DHBsAg) and duck hepatitis B core antigen (DHBcAg) was developed using mononuclear cells from the peripheral blood (PBMC) or spleens (SMC) of immune ducks. Optimal culture conditions for the assay were determined by testing a number of variables, including antigen concentration, cell numbers/well, and the day of harvest. The specificity of the assay was assessed. The assay used 10% pooled duck serum supplement, and 8 x 105 cells/well for PBMC or 5 x 105 cells/well for SMC. The optimum antigen concentration ranged from 0.01 to 0.1 μg/ml for both DHBsAg and DHBcAg. Maximum antigen-specific blastogenesis occurred between 4 to 7 days after establishment of the culture. The use of PHA (10 μg/ml) mitogenesis could predict the optimal cell numbers/well for antigen-specific blastogenesis. The assay demonstrated specific responses by immune ducks compared with those of unexposed ducklings and adult ducks (for DHBsAg P < 0.001; DHBcAg P < 0.05). For immune ducks, PBMC from all 8 ducks responded to DHBsAg, however, cells from only 4 of 7 immune ducks responded to DHBcAg. Splenic mononuclear cells from all immune ducks responded to either DHBsAg or DHBcAg or both antigens.