An inactivated influenza vaccine produced in canine kidney cells (MDCK 33016-PF) contains no egg proteins and may be used to immunize egg-allergic patients. Although no major dog allergens were identified in MDCK 33016-PF cells, minor dog allergens might be present and cause reactions in dog-allergic individuals.To evaluate the allergenicity of the inactivated influenza vaccine produced in cell culture in a mediator release assay.Rat basophil leukemia (RBL) cells transfected with human IgE receptor-1 were sensitized with sera from dog-allergic adults with positive skin prick test reactions to dog extract and detectable dog dander IgE and were stimulated with serial dilutions of vaccine and dog dander extract. N-hexosaminidase release (NHR) was used as a marker of RBL cell degranulation. Western blots were performed, and UniCAP was used to measure dog-specific IgE antibody levels.The median (interquartile range) level of dog dander IgE was 8.31 kU A /L (1.895–14.5 kU A /L) and of dog epithelium IgE was 3.19 kU A /L (0.835–6.27 kU A /L). Median (range) maximum NHR (at the first 10-fold dilution) was 0% (0%–1.4%) to vaccine and 10.2% (0%–35.9%) to dog dander (P < .001). In an egg-allergic control subject, the maximum NHR to a vaccine cultured in chick embryo and containing egg protein was 10.2%. IgE antibodies in pooled sera did not bind to vaccine on immunoblots but produced strong binding to dog dander and epithelium extracts. Serum from an egg-allergic control subject strongly bound embryonated egg-derived vaccine.An influenza vaccine produced in continuous canine kidney cells did not trigger degranulation in RBL cells passively sensitized with human anti–dog IgE.