Humoral immune responses of black-footed penguins (Spheniscus demersus) to DNA-mediated immunization with a β-galactosidase reporter gene expression plasmid were evaluated. Six male and 6 female adult penguins received either test plasmid, pCMV-β, containing the β-galactosidase gene or control plasmid, pCI, lacking a gene for expression. Three birds from each group were used previously in a diluent control group and given one injection of sterile saline. All samples were screened for anti-β-galactosidase antibodies by indirect enzyme-linked immunosorbent assay with anti-chicken immunoglobulin G as secondary antibody. Antibodies to β-galactosidase were detected in the sera of pCMV-β–inoculated penguins, with a peak response on day 21. Antibody titers of the test plasmid group versus both control groups on days 21, 28, and 42 differed significantly. These results demonstrate that black-footed penguins can be safely transfected with the gene encoding β-galactosidase and will mount a humoral response against the in vivo–expressed protein. Knowledge from this initial study can be applied to the development of DNA-mediated vaccines against specific infectious diseases of penguins.